Cip1/Waf1. A panel of eight neuroblastoma cell lines and a TH-MYCN mouse model were investigated for the expression of p21Cip1/Waf1 using RT-qPCR, Western blot, and immunofluorescence. This was followed by investigation of sensitivity towards cisplatin and the p21Cip1/Waf1 inhibitor UC2288. Whereas the cell lines and the mouse model showed low levels of un-phosphorylated p21Cip1/Waf1, the phosphorylated p21Cip1/Waf1 (Thr145) was highly expressed, which in the cell lines correlated to cisplatin resistance. Furthermore, the neuroblastoma cell lines showed high sensitivity to UC2288, and combination treatment with cisplatin resulted in considerably decreased cell viability and delay in regrowth in the two most resistant cell lines, SK-N-DZ and BE(2)-C. Thus, targeting p21Cip1/Waf1 can offer new treatment strategies and subsequently lead to the design of more efficient combination treatments for high-risk neuroblastoma.Platinum-based chemotherapies such as cisplatin are used as first-line treatment for the paediatric tumour neuroblastoma. Although the majority of neuroblastoma tumours respond to therapy, there is a high fraction of high-risk neuroblastoma patients that eventually relapse with increased resistance. Here, we show that one key determinant of cisplatin sensitivity is phosphorylation of the cyclin-dependent kinase inhibitor p21 Neuroblastoma (NB) is the most common extracranial solid tumour in early childhood. It is a heterogenous neoplasia with clinical presentation ranging from spontaneous regression of metastatic disease to a rapidly progressive course , 2. HighIn order to combat development of resistant cells, novel targeted strategies are being implemented for high-risk NB, including small-molecule inhibitors of anaplastic lymphoma kinase (ALK), radiolabelled somatostatin analogues, and monoclonal antibodies against the antigen GD2 , 6. EvenCip1/Waf1 (p21) has been linked to cisplatin resistance in testicular cancer  continues to be the cornerstone of systemic NB treatment. The platinum complex cisplatin is commonly included in first line therapies as adjuvant therapy, with the aim of inducing cytotoxicity and apoptosis , 8, 9. Ur cancer  and in or cancer .The p21 protein is transcriptionally regulated by p53-dependent and –independent pathways and is a key regulator of cell fate. Based on the intracellular localization, the p21 protein shows pleiotropic effects on cell growth and apoptosis in both malignant and non-malignant cells and tissue –16. In tFew small molecule inhibitors of p21, with the aim to inhibit p21 for potential cancer therapy, have been reported so far. These include butyrolactone I , LLW10 , sorafenThe possible role of p21 as an oncogene has previously not been studied in high-risk NB. Therefore, we have here mapped the endogenous expression of p21, both un-phosphorylated and phosphorylated, in a panel of eight high-risk NB cell lines and one NB mouse model. The effect of UC2288, either alone or in combination with cisplatin, was examined to investigate how functional modulation of p21 and p-p21 may alter NB cell viability. Here we report that increased cisplatin resistance was correlated to p-p21, and that combination treatment with UC2288 and cisplatin considerably decreased tumour cell viability. These results suggest an important role for p21 and p-p21 in preventing cisplatin-induced cell death in high-risk NB.TP53 wild type (wt) and five were TP53 mutated (mut) in order to compare between TP53 dependent and independent expression of p21. The non-tumourigenic fibroblast cell line MRC5 and the acute promyelocytic leukemia cell line HL-60 were included as controls (BE(2)-C and Kelly were obtained from ATCC . SK-N-SH, SH-SY5Y, IMR-32, SK-N-AS, SK-N-FI, SK-N-DZ, and MRC5 were a kind gift from professor Per Kogner at Karolinska Institute. HL-60 was a kind gift from Dr. Mohammad Hojjat Farsangi at Karolinska Institute. Eight high-risk NB cell lines were used, where three were TH-MYCN animals were obtained from the Mouse Model of Human Cancer Consortium Repository and kept on a 129X1/SvJ background . We here used tumours from hemizygous mice (MYCN+/−) for immunofluorescent staining for p21 and p-p21 according to the immunofluorescent protocol description.The transgenic ckground . TumoursTwenty-four hours after plating the protein from the NB cell lines, the MRC5, and the HL-60 cell lines were extracted for the baseline data. The protein extraction was done by using RIPA buffer  supplemented with a protease and phosphatase inhibitor  according to the manufacturer’s recommendations, separated electrophoretically and blotted on a nitrocellulose membrane  using standard procedures. All samples were loaded at 20 µg for equal loading. The p21 protein was detected using an anti-p21 antibody  or an anti-p-p21 (T145) antibody , and Cleaved-Caspase3 (C-Casp3) was detected using an anti-C-Casp3 antibody . GAPDH was stained with anti-GAPDH antibody . Protein bands were visualized with secondary antibodies diluted 1:15,000  using a ChemiDoc MP imaging system  or a LI-COR imaging system (LI-COR Biosciences UK Ltd). Analysis of band intensity was performed using ImageJ. All experiments were done in triplicate or quadruplicate, except for the HL-60 experiments which were done in duplicates.® Reverse Transcription manufacturer protocol . RT-qPCR was performed following QuantiText® SYBR® Green PCR manufacturer protocol . The primer sequences used can be found in Each cell line in logarithmic growth was plated onto petri dishes and incubated for 24 hours before samples for RT-qPCR were harvested. mRNA was harvested according to the RNeasy Mini Kit protocol . ß-mercaptoethanol was added at a volume of 10 µl/ml of Buffer RLT. mRNA concentration was measured using Nanodrop™ . Reverse transcription of mRNA into cDNA was performed according to the Quantitect3 in 1xPBS was added and the cells were incubated overnight at +4°C. The primary antibody was then removed, and cells were washed 3x5 min with 1xPBS before the secondary antibody, diluted in TNB buffer, was added. The cells were kept at room temperature for 2 hours before the secondary antibody was removed and the cover slips were washed 3x5 min with 1xPBS. The cells were mounted with Prolong Gold antifade with DAPI  to stain the cell nuclei. A Metafer® Slide Scanning Platform  was used to analyse the cells. The spatial localization of either the p21 or p-p21 expression was done by optical inspection by two independent observers. The mean intensity (mean fluorescence in arbitrary units per cell ± SD) was provided by the Metafersystem software and the low, intermediate, or high expression levels were based on equal range distributions of the values. Following the protocol for immunofluorescence, a triple staining was performed on all eight cell lines using antibodies for p21, the DNA damage marker phosphorylated ataxia-telangiectasia mutated (pATM), and the replication marker Ki67. All experiments were done in triplicate. Antibodies used can be found in Each cell line in logarithmic growth was plated onto round 13 mm #1 coverslips  in petri dishes and incubated for 24 hours before being fixated for 15 minutes using 4% paraformaldehyde . TNB buffer  block was added for 30 min at room temperature. Primary antibody diluted in 0.3% Triton X-100, 0.1% NaN50 , UC2288 at a dose of 10 µM or a combination of cisplatin and UC2288 followed by fixation for 15 min with 4% paraformaldehyde and treated according to the protocol for immunofluorescence or collected for protein according to the protocol for Western blot. All experiments were done in quadruplicates.Twenty-four hours after plating of Kelly and SH-SY5Y, UC2288 at a dose of 10 µM  diluted in dimethyl sulfoxide (DMSO) was added for 2 hours, 4 hours or 6 hours before the cells were fixated for 15 min with 4% paraformaldehyde and treated according to the protocol for immunofluorescence, or collected for protein or mRNA according to the protocol for either Western blot or RT-qPCR. UC2288 was added at a concentration of 10 µM based on the literature . All exp2. Seventy-two hours after treatment, 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was performed where 20 µl CellTiter 96® Aqueous One Solution Assay  was added to each well. Plates were incubated for 2.5 hours and metabolic activity was analysed in the microplate reader FLUOstar Omega  by measuring absorbance at 490 nm and 690 nm. All treatments were done in triplicate.Dose–response curves for cell viability were obtained after treatment for 72 hours with either UC2288 or cisplatin, in eight NB cell lines and one fibroblast cell line, MRC5, which was used as a non-tumourigenic control for drug toxicity. UC2288 was added as a single agent at concentrations of 50 µM, 10 µM, 5 µM, 1 µM, 0.5 µM, 0.1 µM, 0.05 µM or 0.01 µM and cisplatin at concentrations of 100 µM, 50 µM, 10 µM, 5 µM, 1 µM, 0.5 µM, 0.1 µM or 0.05 µM. For the combination treatments, cisplatin was added at concentrations of 100 µM, 50 µM, 10 µM, 5 µM, 1 µM, 0.5 µM, 0.1 µM or 0.05 µM either alone or in combination with UC2288 at a concentration of either 10 µM or 1 µM. The plates were incubated at 37°C, 5% CO3 cells), Kelly (10 × 103 cells), SK-N-DZ (10 × 103 cells) and BE(2)-C (5 × 103 cells) were seeded in 200 µl medium/well in a 96 well plate, and the edges were filled with medium to avoid edge effects. Twenty-four hours after seeding each cell line was treated with the corresponding IC50 value for cisplatin and 10 µM UC2288 for 48 hours. DMSO (mock) was used as negative controls. The plates were scanned by an IncuCyte S3 Live® Cell Analysis System  48 hours post treatment followed by medium change. The plates were thereafter scanned followed by medium change every 96 hours for a total of 30 days. The proliferation was determined by measuring the cell confluency. Once the cell lines reached 100% confluency, they were maintained for the duration of the experiment. Each treatment was done in 5-10 replicates and the data are presented as mean ± Standard Error of the Mean (SEM).SK-N-FI . In general, a CI of <1 is considered a positive and a CI of >1 a negative combined effect. More specifically, CI<0.70 was defined as synergy, CI>1.45 as antagonism, and values in between as additive effects, according to the recommendations of the ComboSyn software.To estimate the effects of the combination treatments the median-effect method of Chou   was appl50 value for each of the single treatments or for the combination treatment. The data was logarithmically transformed and normalized against 0% (no cell viability) and 100% (maximum cell viability) controls for the combination treatments. For determining IC50 values for the single treatments, the nonlinear regression analysis method log(inhibitor) vs normalized response was used. For determining IC50 values for combination treatment, the nonlinear regression analysis log(inhibitor) vs normalized response – variable slope was used. Linear regression analysis with Pearson’s correlation coefficient was used to investigate a possible relationship between the IC50 values of UC2288 and cisplatin, and between the fraction of p21 positive cells with either UC2288 or cisplatin. All analyses were performed in Graphpad Prism (version 9.3.1). Western blot data are presented as mean ± standard deviation (SD). Immunofluorescence data were analysed in RStudio and the percentage of positive cells followed by the mean ± standard deviation (SD) were calculated for each combination. Data was analysed using Student’s t-test or one-way ANOVA with Tukey post hoc test, as indicated in the text. Graphpad Prism (version 9.1.0) was used for generation of graphs.Following the MTS-assay, the data was analysed to obtain the ICTP53 wt cell lines or TP53 mut cell lines showed that among the TP53 wt cell lines SK-N-SH had the highest p21 mRNA expression followed by IMR-32 and among the TP53 mut cell lines SK-N-FI and Kelly showed the highest p21 mRNA expression  mRNA expression level of p21 (CDKN1A) within eight high-risk NB cell lines and one non-tumourigenic fibroblast cell line, MRC5. The MRC5 cell line had the highest p21 mRNA expression compared to the remaining cell lines (p<0.0001)  among all the tested cell lines  and IMR-32 , compared to SH-SY5Y (4.7% ± 0.9). In the TP53 mut cell lines, the fraction of p21 positive cells was generally lower with SK-N-FI  having a significantly higher fraction of positive cells compare to BE(2)-C (1.5% ± 0.3) and SK-N-DZ (0.1% ± 0.1). Cells positive for p21 were also detected in SK-N-AS (5.7% ± 0.6) and Kelly (4.9% ± 0.5)  and IMR-32 (2.1% ± 2.2). Among the TP53 mut cell lines, SK-N-FI (93.9% ± 9.1) and SK-N-DZ (97.6% ± 2.2) had significantly higher fractions of p-p21 positive cells compared to SK-N-AS, Kelly, and BE(2)-C cell lines which had moderate expression   compared to the three cell lines with the highest expression, SH-SY5Y (78.6% ± 17), SK-N-FI (93.9% ± 9.1), and SK-N-DZ (97.6% ± 2.2) (https://www.proteinatlas.org/ENSG00000124762-CDKN1A/cell+line). No p21 or p-p21 staining was detected in the HL-60 cell line when investigated with Western blot and immunofluorescence validating the specificity of the antibodies -C, which displayed high expression. Moderate cytoplasmic expression was observed in SK-N-SH, SK-N-FI, and SK-N-AS, followed by low or no detectable cytoplasmic staining in IMR-32, SH-SY5Y, Kelly, and SK-N-DZ , and the mice spontaneously develop NB at 5.6–19 weeks of age  and IMR-32  showed higher fraction of positive cells compared to SH-SY5Y (2.7% ± 0.4). Among the TP53 mut cell lines SK-N-FI (9.3% ± 0.8) had the highest fraction of cells expressing low levels of p21 followed by SK-N-AS (5.3% ± 0.5), Kelly (4.8% ± 0.5), BE(2)-C (1.2% ± 0.2), and SK-N-DZ (0.1% ± 0.1)  having a significantly higher fraction of positive cells compared to SH-SY5Y (0.2% ± 0.2). There was only one TP53 mut cell line with cells positive for high p21 expression, SK-N-FI (0.1% ± 0.1). The remaining TP53 mut cell lines SK-N-AS, Kelly, BE(2)-C, and SK-N-DZ showed no cells with high p21 expression , and the proliferation marker Ki67. First, each marker was analyzed on its own with the fraction of p21 positive cells being higher in Analysis of co-expression of the markers showed that within each cell line, a very small fraction of p21 expressing cells displayed co-expression of p21 and pATM (0-0.2%)  to 32.0 µM for BE(2)-C -C, displaying the highest IC50 values for cisplatin, were selected for further investigation of the cisplatin response. Each of the four cell lines were treated with the corresponding cisplatin IC50 concentration for 24 hours following analysis, using immunofluorescence, of DNA damage (pATM) and proliferation (Ki67) as a predictor of treatment outcome -C, had a decrease either in the fraction or the mean intensity of p21 positive cells. At the same time, three of the four tested cell lines, all but SK-N-FI, had an increase in either the fraction or the mean intensity of p-p21 positive cells, indicating that there is a preference of p-p21 activation, compared to p21, following cisplatin treatment.Taken together, the 50 values ranging between 4.3 µM to 53.9 µM -C. Regrowth (confluency) was mapped during a period of 30 days following a pulse treatment for 48 hours of mock or UC2288 at three different concentrations: 1 µM, 10 µM or the corresponding IC50 value for each cell line. SK-N-FI showed the highest sensitivity to the treatment, with a delay in growth at all three tested concentrations of UC2288 (50 dosing of UC2288 before an increase in confluency (regrowth) was observed  also had low IC50 values for cisplatin. On the other hand, BE(2)-C and SK-N-DZ had higher IC50 values for both UC2288 and cisplatin, implying that they are more resistant to single agent treatment with these drugs  for SK-N-FI. For BE(2)-C, the cisplatin IC50 value shifted from 29.1 µM to 40.4 nM and for SK-N-DZ from 30.2 µM to 31.2 nM  when combined with UC2288 at 10 µM. Moreover, a small shift in the cisplatin IC50 value was observed when combined with following UC2288 at 1 µM -C and SK-N-DZ. Due to the huge shift following combination treatment with UC2288 at 10 µM, the IC50, UC2288 10 µM, or combination of cisplatin IC50 and UC2288 10 µM for each corresponding cell line. Two cell lines, SK-N-DZ and BE(2)-C, showed delay in growth following combination treatment compared to a single cisplatin treatment  was mapped during a period of 30 days following a pulse treatment for 48 hours of mock, cisplatin ICTaken together, combination treatment with UC2288 and cisplatin reduced viability in both sensitive, Kelly and SK-N-FI, and resistant, BE(2)-C and SK-N-DZ, cell lines, even though the effect was more profound in the sensitive cell lines. Furthermore, combination treatment showed increase in cell death, indicated by C-Casp3, and a delay in regrowth compared to a single treatment with cisplatin in the two resistant cell lines, BE(2)-C and SK-N-DZ.TP53 wt and TP53 mut cell lines showed expression. However, there was discrepancies between the expression level of p21 mRNA and the p21 protein, both as un-phosphorylated and phosphorylated protein. This is in concordance with studies showing general poor correlation between expression levels of mRNA and protein -C, have MYCN amplification. The clinical prevalence of these genetic alterations is seen among 20-30% of NB tumours which have MYCN amplification, and TP53 mut are observed in a high proportion following relapse, indicating a mechanistic relevance in the development of therapy resistance -C cell line where a reduction of cytoplasmic p-p21 was observed when analyzed with Western blot but not when analyzed with immunofluorescence which detects both cytoplasmic and nuclear p-p21 expression, further validating the ability of UC2288 to inhibit mainly cytoplasmic p-p21. Further treatment of TP53 mut NB cell lines with UC2288 in combination with cisplatin showed a synergistic effect and a dramatically reduced the IC50 value, with an increase in cell death, indicated by C-Casp3 activation, suggesting UC2288´s potential as a drug against chemotherapy resistance .SSF contributed to conceptualization, data curation, methodology, project administration, resources, software, supervision, visualization, and funding acquisition. AS, VH and SSF performed the formal analysis, statistical analysis, investigation, and validation. AS, MW and SSF wrote the first draft of the manuscript. MW and SSF wrote sections of the manuscript. All authors contributed to manuscript revision, read, and approved the submitted version.This research was funded by the Childhood Cancer Foundation grant numbers, TJ2019-0118, PR2019-0101, and PR2021-0037, the Swedish Cancer Society grant number 21-0313 SIA. Eva and Oscar Ahrén Foundation, Stockholm, and Magnus Bergvalls stiftelse grant number 2021-04262.The authors wish to thank Dr. Per Kogner for NB cell lines and Dr. Mohammad Hojjat Farsangi for the HL-60 cell line. We would also like to thank Dr. Ingrid Lilienthal and Hala Ibrahim Mohammad Habash for scientific discussions and technical assistance.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Non-small cell lung cancer (NSCLC) patients acquire resistance to tyrosine kinase inhibitors (TKIs) via EGFR mutations or overexpression of vascular endothelial growth factor receptor-2 (VEGFR-2). In this study, we elucidated the mechanism of EGFR-TKI resistance mediated by VEGF/VEGFR in EGFR-mutated NSCLC cell lines and Erlotinib-resistant cell lines as compared to parental cell lines. Increased expression of VEGF, VEGFR-2, and NP1 was observed in Erlotinib-resistant cell lines. Furthermore, we observed an increased efficacy of Erlotinib in combination with a VEGFR-2 inhibitor in Erlotinib-resistant cell lines. Late-stage NSCLC patients with high expression of VEGFR-2 had shorter survival times compared to patients with low VEGFR-2 expression. These results indicate that VEGFR-2 may play a key role in EGFR-TKI resistance that can be overcome with a combination treatment of Erlotinib and a VEGFR-2 inhibitor, which may serve as an effective treatment option for NSCLC patients with EGFR mutations.NSCLC treatment includes targeting of EGFR with tyrosine kinase inhibitors (TKIs) such as Erlotinib; however, resistance to TKIs is commonly acquired through T790M EGFR mutations or overexpression of vascular endothelial growth factor receptor-2 (VEGFR-2). We investigated the mechanisms of EGFR-TKI resistance in NSCLC cell lines with EGFR mutations or acquired resistance to Erlotinib. These studies showed upregulated gene and protein expression of VEGF, VEGFR-2, and a VEGF co-receptor neuropilin-1 (NP-1) in Erlotinib-resistant (1.4–5.3-fold) and EGFR double-mutant  NSCLC cells compared to parental and EGFR single-mutant (L858R) NSCLC cell lines, respectively. Immunofluorescence and FACS analysis revealed increased expression of VEGFR-2 and NP-1 in EGFR-TKI-resistant cell lines compared to TKI-sensitive cell lines. Cell proliferation assays showed that treatment with a VEGFR-2 inhibitor combined with Erlotinib lowered cell survival in EGFR double-mutant NSCLC cells to 9% compared to 72% after treatment with Erlotinib alone. Furthermore, Kaplan–Meier analysis revealed shorter median survival in late-stage NSCLC patients with high vs. low VEGFR-2 expression (14 mos vs. 21 mos). The results indicate that VEGFR-2 may play a key role in EGFR-TKI resistance and that combined treatment of Erlotinib with a VEGFR-2 inhibitor may serve as an effective therapy in NSCLC patients with EGFR mutations. Lung cancer is the leading cause of cancer-related deaths. It was projected that, in 2021, 235,760 new lung cancer cases would be diagnosed and that 131,880 individuals would die from the disease in the United States alone . Among tEGFR is a receptor tyrosine kinase (RTK) overexpressed in 40–80% of NSCLC patients. When dysregulated, EGFR facilitates tumorigenic signaling ,6. ExpreNumerous tyrosine kinase inhibitors (TKIs) have been developed to act against activating mutations in EGFR. First-generation TKIs such as Erlotinib and second-generation TKIs such as Afatinib are used to overcome EGFR aberrant signaling by covalently binding to EGFR’s kinase domain . The devAs observed in lung cancer, dysregulation of the EGFR pathway results in upregulation of the human vascular endothelial growth factor (VEGF) pathway. Previous studies using cell culture models have shown that EGFR activation via epidermal growth factor (EGF) or transforming growth factor alpha (TGF-α) increases the production of VEGF ,16. VascNeuropilins (NPs)  are non-tyrosine kinase receptors that act as VEGF co-receptors. Prior studies have shown that co-expression of NPs is significantly correlated with poor prognosis and tumor progression in NSCLC . NeuropiTo better understand the mechanism of EGFR-TKI resistance in NSCLC and to develop more effective therapies against it, we studied three biomarkers linked to increased angiogenesis in NSCLC . We inveErlotinib hydrochloride (N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy) quinazolin-4-amine; C22H23N3O4•HCl) was obtained from LC laboratories , reconstituted in Dimethyl Sulfoxide (DMSO) at a concentration of 20 mM, and then stored in aliquots of 20 µL at −20 °C. ZM 323,881 hydrochloride (5-((7-Benzyloxyquinazolin-4-yl)amino)-4-fluoro-2-methylphenol hydrochloride), a selective inhibitor of VEGFR2, was obtained from Tocris Bioscience, a Bio-Techne brand  , reconstRabbit polyclonal antibody for VEGFR-2 (cat. no. SAB4501645-100UG) was obtained from Sigma-Aldrich. Rabbit polyclonal antibody for VEGFR-2 (cat. no. 07-716-I) was obtained from Millipore . Rabbit mouse monoclonal neuropilin-1 antibody (cat. no. Ab81321) was obtained from Abcam . Goat polyclonal antibody for neuropilin-1 (cat. no. sc-7239) was obtained from Santa Cruz . Mouse monoclonal VEGF (C-1) antibody (cat. no. sc-7269) was obtained from Santa Cruz . Bevacizumab, a humanized monoclonal VEGF-165 antibody (cat. no. NBP2-59637), was obtained from Novus Biologicals . Human antibodies CD309 (VEGFR-2/KDR)-APC (cat. no. 130-098-910) and CD304-PE (BDCA-4/Neuropilin-1) (cat. no. 130-098-876) were obtained from MACS Miltenyi Biotec . All primary antibodies were diluted according to the manufacturer’s instructions in TBST with 1% BSA. Secondary antibodies were prepared at a dilution of 1:1000 in TBST with 1% blocking grade milk (cat. no. 170-6404XTU) from Bio-Rad .2 and cultured according to ATCC’s instructions. H2170 and H358 cell lines have wild-type EGFR status, whereas H3255 (L858R) and H1975 (L858R and T790M) cell lines have mutated EGFR, as documented by ATCC. H2170 and H358 cell lines were cultured in increasing drug concentrations of Erlotinib to obtain EGFR-TKI-resistant cells. H2170-ER and H358-ER cells were obtained and cultured in the presence of erlotinib, as described previously [H2170, H358, H3255, and H1975 NSCLC cell lines  were purchased from American Type Culture Collection (ATCC) . Cell lines were grown in incubators at 37 °C with 6% CO(−Δ(ΔCt)) was calculated to evaluate the fold changes in gene expression. All primers  and H358-P/ER cells were plated in 35 mm petri dishes and allowed to adhere and grow for 24 h. Cells were then starved for 24 h and total RNA was collected using a PureLink RNA Mini Kit (cat. no. 12183018A) from Life Technologies, according to the manufacturer’s protocol. RNA was quantified using the Take3 NanoDrop method. Total RNA was used to perform qPCR with an Applied Biosystems 7300 Real Time-PCR system. For this procedure, a SuperScript™ III Platinum™ SYBR™ Green One-Step qRT-PCR Kit (cat. no. 11736059) from Life Technologies was employed and the manufacturer’s protocol was followed. The cycle threshold (Ct) values were recorded and normalized using GAPDH, and 2 primers  used forNSCLC cells were cultured as described previously . H2170-ETM 488 (1:250)  and Hoechst nuclear staining dye   diluted in 1X PBS with 1% BSA. Slides were then mounted and covered with a coverslip using fluorogel . Stained cells were observed using an Olympus Fv10i Fluoview Confocal Microscope and images were captured. Average fluorescence intensity of staining was quantitatively measured using Olympus Fluoview image analysis software.NSCLC cells were seeded on an eight-well chamber slide, fixed, permeabilized, blocked, and incubated at 4 °C overnight with primary antibodies for VEGFR-2 and NP-1. Primary antibodies were then removed and the cells were incubated in the dark at room temperature for an hour with anti-mouse/rabbit IgG secondary antibody conjugated with DyLightNSCLC cells were seeded in a 100 mm petri dish. After 48 h, the growth medium was removed. The cells were then washed with PBS and treated with Accutase Cell Detachment Solution  in an incubator for five minutes or until all of the cells were detached. Cells were collected in a 15 mL tube, pelleted using a centrifuge, and then re-suspended in flow cytometry buffer (PBS + 2 mM EDTA + 0.5% BSA). Cells were pelleted again and probed with fluorophore-labeled antibody for ten minutes on ice. The cells were washed with flow cytometry buffer to remove excess antibody, then suspended in FACS buffer for analysis with a BD FACS CALIBUR flow cytometer.NSCLC cells were seeded on a 96-well plate in RPMI medium with 10% FBS. After 24 h, cells were treated with TKIs, suspended in 100 µL starving media (RPMI + 0.5% BSA) per well, and incubated for an additional 72 h at 37 °C. Cells were then washed with RPMI media without phenol red and 100 µL of phenol red-free RPMI media was added to the cells. Subsequently, 10 µL of MTT reagent  was added to each well and plates were incubated for four hours at 37 °C. Finally, 100 µL of MTT solubilization reagent (acidic isopropanol) was added to each well and mixed to dissolve any and all formazan crystals formed. Absorbance was measured using a spectrophotometer at wavelengths of 570 nm and 630 nm, and percent cell viability was calculated with respect to controls. This experiment was performed in replicates of six for each treatment condition.2 vented flasks and allowed to adhere for 24 h in RPMI medium with 10% FBS. Medium was then removed, followed by two 1X PBS washes. Serum-free medium was added and incubated for 24 h. Then, the medium was pipetted into a micro-centrifuge tube and centrifuged for ten minutes at 4 °C with a LABNET Refrigerated Z326K centrifuge  at 1500 rpm. The supernatant was immediately aliquoted, avoiding the debris pelleted, and samples were stored at −70 °C. Collection of conditioned medium was repeated for 24 and 48 h time points. The commercial ELISA kit Human VEGF-A ELISA from RayBiotech  was used to detect and quantify the amount of VEGF in the NSCLC cell lines at two separate time points: 24 h and 48 h. The vendor’s protocol was followed to run the experiment, and each test was run in triplicate.NSCLC cells were plated in 25 cmWe analyzed 79 paraffin-embedded, formalin-fixed tissues from patients with NSCLC. All cases were retrieved from the pathology archives at Rockford Memorial Hospital and the Swedish American Hospital with institutional approval, in accordance with the Institutional Review Board protocol (351597-11 and 17 August 2018). Immunostaining procedures were conducted as described previously . Approprt-test was used to analyze the statistical significance of the data. A p-value of less than 0.05 was considered statistically significant throughout the study.All experiments were performed three to five times. The Student’s The gene expressions of potential EGFR-TKI-resistance modulators were analyzed in the wild type-EGFR NSCLC cell lines H358-P and H2170-P and compared to corresponding Erlotinib-resistant H358-ER and H2170-ER cell lines. H358 cell lines also possess a unique KRAS mutation. The results demonstrated an increase in gene expression of VEGF (2.5-fold), VEGFR-2 (3.5-fold), and NP-1 (1.4-fold) in H2170-ER compared to H2170-P cells, as can be seen in Immunoblotting studies were performed to assess the modulation of VEGFR-2 and NP-1 protein expression in wild-type EGFR NSCLC cells (H2170-P/ER and H358-P/ER), TKI-sensitive EGFR L858R-mutant NSCLC cells (H3255), and TKI-resistant EGFR L858R- and T790M-mutant NSCLC cells (H1975). Each cell line was analyzed without treatment, after treatment with Erlotinib, after treatment with epidermal growth factor (EGF), and after treatment with a combination of Erlotinib and EGF A.The expressions of VEGFR-2 and NP-1 from Western blots were quantified and analyzed. The results demonstrated an increase in VEGFR-2 (3.6-fold) and NP-1 (2.8-fold) expression in the presence of 10 µM Erlotinib in H2170-ER cells compared to the H2170-P cell line subjected to the same treatment B. AdditiAfter observing upregulation of gene and protein expression of VEGFR-2 and NP-1 in ER cells, the levels of expression of these proteins were measured on cell surfaces. Flow cytometry assays were performed to measure the expression of VEGFR-2 and NP-1 in H2170-P/ER, H358-P/ER, H3255, and H1975 cell lines. In H2170-ER cells as compared to H2170-P cells, there was an increase in mean fluorescent intensity for VEGFR-2 (2.1-fold) and NP-1 (3.8-fold) A. In H35Immunofluorescence (IF) was performed to confirm and further study the specific localization of VEGFR-2 and NP-1 in parental (H2170-P and H358-P), Erlotinib-resistant (H2170-ER and H358-ER), EGFR single-mutated (H3255), and EGFR double-mutated (H1975) cell lines. In H2170-ER cells, VEGFR-2 was expressed mainly on the cell surface and in the cytoplasm, while in H2170-P cells, VEGFR-2 was expressed mainly in the cytoplasm A. In H21In H358-ER cells, VEGFR-2 was expressed on the cell surface, in the cytoplasm, and in the nucleus, while in H358-P cells, VEGFR-2 was minimally expressed on the cell surface, in the nucleus, and in the cytoplasm B. NP-1 wIn H1975 cells, VEGFR-2 was expressed on the cell surface, in the cytoplasm, and in the nucleus, while VEGFR-2 was minimally expressed in the nucleus and on the membrane of H3255 cells C. NP-1 wAn enzyme-linked immunosorbent assay (ELISA) was performed to study the role of VEGF secretion in angiogenesis and EGFR-TKI resistance. Human VEGF concentration was quantified and analyzed for all cell lines and used to calculate levels of VEGF secretion. The results demonstrated that VEGF concentration (pg/mL) increased from 24 h to 48 h in all cell lines studied . We obseTo further study the effect of VEGF in EGFR-TKI resistance, we investigated the use of a humanized monoclonal VEGF antibody, bevacizumab (Avastin), which binds to and neutralizes VEGF. An MTT cell viability assay was performed to assess the effects of Avastin, Erlotinib, and both in combination in Erlotinib-resistant and EGFR-mutated NSCLC cells lines. Cells being resistant to 10 µM of Erlotinib, this was the concentration that was used, and since Avastin showed a similar inhibitory effect on cells in a concentration range of 200 ng/mL to 1000 ng/mL, we used 200 ng/mL of Avastin. The 10 µM Erlotinib treatment decreased cell viability to between 62 and 82% compared to the diluent, while the 200 ng/mL Avastin treatment decreased cell viability to between 84 and 89% compared to the diluent . FurtherAfter studying the effects of treatment against VEGF, we studied the inhibition of VEGFR-2 in Erlotinib-resistant and EGFR-mutated NSCLC cell lines to further elucidate its role in EGFR-TKI resistance. An MTT cell viability assay was performed with 5 µM Erlotinib treatment, 12 µM ZM 323–881 HCl (ZM) (VEGFR-2 inhibitor) treatment, and a combination treatment of Erlotinib and ZM. We tried different concentrations of ZM and selected a concentration of ZM which resulted in less than 50% inhibition. We used Erlotinib at a concentration of 5 uM, since the resistant cell lines studied showed 25–26% inhibition with 5 μM Erlotinib. We wanted to study a combination effect, so we kept the concentration of Erlotinib at half the dose we used for other aspects of the studies. As seen in p < 0.05) (Immunohistochemistry (IHC) was performed to analyze the expression of VEGFR-2 in tumor tissues from NSCLC patients. Forty-eight NSCLC tumor tissues were assessed for VEGFR-2 expression through IHC with VEGFR-2 staining A. Kaplan < 0.05) B.In this study, we recorded upregulation of the gene expression of VEGF, VEGFR-2, and NP-1 in Erlotinib-resistant cell lines as compared to parental cells and in EGFR double-mutant cells as compared to their single-mutant counterparts . In all VEGFR-2 and NP-1 protein expressions were also increased in EGFR double-mutant H1975 cells compared to H2170-ER and H358-ER cell lines, a result attributed to the T790M mutation in H1975 cells B. UnlikeThese mutations cause altered expression of EGFR, which results in the activation of downstream signaling pathways, such as the MAPK, FAK/Paxillin, and PI3K/AKT/mTOR pathways, which, in turn, promotes tumor cell proliferation, survival, migration, and angiogenesis .AccordinStudies have shown that NP-1 interacts with VEGFR-2 in a cis position, which, in turn, increases the binding affinity of VEGF to VEGFR-2 . In thisOur results also demonstrated that total VEGF secretion increased over time . VEGF seAfter determining that VEGF inhibition is not useful for overcoming EGFR-TKI resistance, we inhibited VEGFR-2 using a selective VEGFR-2 inhibitor, ZM 323–881 HCl (ZM). Based on our results, treatment with ZM alone and treatment with a combination of Erlotinib and ZM both significantly decreased cell viability in Erlotinib-resistant and EGFR-mutated cell lines . These rDuring NSCLC tumor screening with VEGFR-2 and NP-1 by IHC assays, we studied the expression levels of these angiogenic biomarkers in late- and early-stage NSCLC patients. VEGFR-2 is a transmembrane tyrosine kinase receptor, and studies have shown that VEGFR-2 is internalized with the help of NP-1 . Our floThis study investigated three angiogenic biomarkers, VEGF, VEGFR-2, and NP-1, that may play a role in EGFR-TKI resistance. In EGFR-TKI-resistant cells compared to TKI-sensitive cell lines, an upregulation of VEGFR-2 and NP-1 gene and protein expression, as well as increased cell surface expression, was observed. In addition, we observed that combinatorial treatment of Erlotinib and a selective VEGFR-2 inhibitor may serve as a more effective treatment option than inhibition of VEGF with a humanized monoclonal antibody Avastin. TKIs such as Erlotinib and Gefitinib have no effect on lung adenocarcinoma patients with KRAS mutations; however, combination therapy with Erlotinib and VEGFR-2 inhibitor can help achieve better prognoses for these patients. Our studies suggested that high VEGFR-2 expression is associated with poor prognosis in NSCLC patients, and we also studied single- and double-mutant NSCLC cell lines to provide a new outlook on ways to overcome EGFR-TKI resistance in NSCLC patients.


Soluble α-Klotho (s-Klotho) is a circulating protein with pleiotropic effects that mainly induce protective effects. Our study investigates the associations between s-Klotho and several established inflammatory biomarkers, with the aim of examining whether s-Klotho levels are representative of inflammatory states.A total of 11,128 eligible participants from NHANES 2007–2016 were included in our study. Levels of four inflammatory biomarkers, uric acid (UA), C-reactive protein (CRP), white blood cell (WBC) count, and mean platelet volume (MPV), were examined for their relationship with s-Klotho levels. Sub-analyses sorted the total population by gender and into four quartiles. Linear regression models were used to evaluate the strengths of associations.p<.001), and this relationship was stronger in women than in men . In addition, all four biomarkers manifested stronger associations with s-Klotho in higher quartiles, and the highest absolute values of β coefficients appeared in Q4 vs. Q1.All four inflammatory biomarkers were significantly associated with s-Klotho levels. UA, CRP, and WBC count showed an inverse association, while MPV showed a direct one. Of the four markers, UA was most strongly correlated with s-Klotho levels  levels are significantly associated with the inflammatory markers uric acid, C-reactive protein, white blood cell count, and mean platelet volume.S-Klotho is involved in inflammatory processes and plays a protective role.S-Klotho may serve as an inverse indicator of inflammation.s-Klotho is significantly associated with well-recognized inflammatory biomarkers. A decrease in s-Klotho levels implies a general inflammatory status; therefore, s-Klotho serves as a potential biomarker that is inversely correlated with inflammatory conditions. Further applications in clinical practice will provide us with a better understanding of its role. In addienotypes ,6, endotenotypes .2+ transient receptor potential vanilloid type 5 in nephrons, which contributes to the maintenance of normal renal function [Emerging evidence illustrates a protective role of s-Klotho in various disorders, prompting researchers to investigate its underlying mechanisms. A study demonstrated that s-Klotho inhibits the insulin/insulin-like growth factor-1 signalling pathway, which promotes resistance to oxidative stress, thereby extending the lifespans of mice . Other sfunction ,13.Because many studies have focussed on the benefits of s-Klotho in individual organs, there is less known about s-Klotho as a circulating factor and how it affects systemic health. This study explored the relationship between established inflammatory biomarkers and s-Klotho levels, aiming to determine whether s-Klotho modulates inflammation generally and its relationship with separate biomarkers.The National Health and Nutrition Examination Survey (NHANES) 2007–2016  databaseWe collected information from NHANES 2007–2016 because the main biomarker of interest, s-Klotho, is available from these years. There were 40,617 participants between 2007 and 2016, and we initially included those with complete data about s-Klotho levels. Subsequently, we excluded those with missing data on inflammatory biomarkers, including serum levels of uric acid (UA) and C-reactive protein (CRP), white blood cell (WBC) count, and mean platelet volume (MPV), leaving 11,128 subjects eligible for further analyses. We performed analyses in the total cohort and in men and women separately to evaluate gender differences. In addition, we divided the values of inflammatory biomarkers into quartiles for detailed comparisons. A flowchart showing the design and structure of our study is presented in All details about laboratory methodology and protocols are provided in the NHANES Laboratory/Medical Technologists Procedures Manual . s-KlothCRP concentration data included CRP values from 1999–2010 and high-sensitivity CRP from 2015–2018. The former values were determined using latex-enhanced nephelometry, and the latter was based on the highly sensitive near-infrared particle immunoassay rate methodology. WBC counts and MPVs are included in complete blood count tests. They were both analysed on the Beckman Coulter UniCel DxH 800 . Albumin levels were measured using the DxC800 as a bichromatic digital endpoint method. High-density lipoprotein cholesterol levels were examined enzymatically with a Roche/Hitachi Modular P Chemistry Analyser  according to the manual.2 (weight in kilograms/height in meters). The following categories for race were used: Mexican–American, other Hispanic, non-Hispanic White, non-Hispanic Black, and other races. A history of smoking was defined as having smoked at least 100 cigarettes in life. Histories of medical conditions were confirmed by asking the participants if they had ever been told by a doctor that they had one of the following conditions: coronary heart disease, angina, diabetes, and hypertension.Demographic information was acquired by trained examiners in household interviews. The formula for body mass index was kg/mp values<.05 were considered statistically significant. Linear regression analyses were utilized, and β coefficients were calculated to estimate the strengths of relationships. Three models were provided for each analysis to eliminate the effects of nuisance variables: Model 1 = unadjusted; Model 2 = adjusted for age, gender, body mass index, and race/ethnicity; Model 3 = Model 2 + adjusted for history of angina, coronary heart disease, diabetes mellitus, hypertension, and smoking status.SPSS  was used for statistical analyses. Qualitative data were expressed in percentages (%), while quantitative variables were expressed as means and standard deviations. All 3 cells/μl for WBC count, 862.29 ± 306.37 pg/ml for s-Klotho, 5.54 ± 1.43 mg/dl for UA, 0.46 ± 0.89 mg/dl for CRP, and 8.11 ± 0.95 fl for MPV.This study included 11,128 participants, including 5402 (48.5%) men and 5726 (51.5%) women. Demographic and laboratory profiles are shown in p<.001). The remaining variables had the following β coefficients in descending order: MPV ; CRP ; and WBC count . Further analyses showed gender differences for each biomarker. In women, UA and WBC count were more strongly correlated with s-Klotho levels  ). In men, CRP and MPV were more strongly correlated with s-Klotho levels  ).As shown in p<.001; Q3 vs. Q1, β coefficient= −65.72, 95% CI= −82.32, −49.13, p<.001; Q4 vs. Q1, β coefficient= −107.13, 95% CI= −123.78, −90.48, p<.001).To determine whether the associations between s-Klotho and the four inflammatory biomarkers changed on a gradient, we stratified levels of the four biomarkers into quartiles. In Our study indicates that several established and widely used inflammatory biomarkers are associated with s-Klotho levels. We demonstrated that s-Klotho functions as an inverse indicator of inflammation because of its inverse association with UA, CRP, and WBC count, and its direct association with MPV. Among these, UA was most strongly correlated with s-Klotho. These findings suggest that s-Klotho may serve as a predictive biomarker for inflammation.p=.022) [Few studies have mentioned the relationship between s-Klotho and inflammatory cytokines. A case–control study with a designated experimental group of 110 patients with histories of cardiovascular disease (CVD) showed that s-Klotho levels were significantly lower in the CVD group and inversely correlated with CRP and TNFα/IL10 . Anotherp=.022) . Howevervia the ROS–MAPK–NF-κB signalling pathway in rat vascular smooth muscle cells, which contributes to the development of CVD [Hyperuricaemia has long been associated with gout , but rect of CVD . In contt of CVD . The medt of CVD . Neverthp = .13). In our study, the inverse association remained significant in all models, including both the unadjusted and fully adjusted ones. This indicated that even after taking the following factors into consideration—age [The inverse relationship between CRP and s-Klotho has been described in CVD patients in a study performed in Santa Cruz de Tenerife, Spain . Howevertion—age , historytion—age —which alElevated WBC count, or leukocytosis, is a clinical sign often linked with infection or inflammation . MPV is et al. reported that under hyperglycaemic conditions, the shedding of Klotho is expedited, causing a decrease in membrane-bound Klotho and an increase in s-Klotho. They also demonstrated the addition of Klotho to cell medium improved kidney function via effects such as increased glycolytic capacity and lowered albumin permeability. Collectively, their findings supported the idea that Klotho ameliorates inflammation, although how s-Klotho predicts inflammatory status in diabetic nephropathy differed from our results. In view of this, the interpretation of serum s-Klotho levels in specific diseases requires further investigation.Despite the general negative relationship between s-Klotho levels and inflammatory biomarkers, under certain circumstances, the association may be positive. A recent study measuring serum and urine s-Klotho levels in rats found significantly higher serum levels of s-Klotho in diabetic subjects . InitialLevels of biomarkers discussed in the present study were altered significantly during the inflammatory process; however, each was regulated in by a unique mechanism. Elevation in circulating WBCs indicates the initiation of host defences against various kinds of injuries, and the five types of WBCs, neutrophils, lymphocytes, eosinophils, basophils, and monocytes, share general anti-inflammatory properties, although each mediates specific immune responses . IncreasNumerous studies that have investigated the functions of Klotho have demonstrated its distinctive role in vascular homeostasis. In CVD, Klotho suppresses IL-6 production in endothelial cells , inhibitComparing the four biomarkers led to the noteworthy finding that UA levels had the strongest relationship with s-Klotho levels, which may be attributable to their shared site of production—the kidney. Serum UA level is determined mainly by reabsorption and excretion in the kidney , and s-KThe pros and cons of incorporating s-Klotho testing into routine clinical practice are worth discussing. Compared with UA, CRP, WBC count, and MPV, which are already commonly used laboratory markers, the importance of Klotho has only been determined in the past decade. While the other four markers are altered in most inflammatory conditions, s-Klotho stands out because it is regulated in particular organs, such as the kidneys, and in cardiovascular-involved diseases, which permits it to provide distinct diagnostic values. However, Klotho is more costly to determine than established biomarkers, which may limit its application in general practice at the present time.The findings of this study must be viewed considering some limitations. First, our study revealed a positive relationship between s-Klotho and MPV, illustrating that decreased MPV is linked to inflammation. However, studies have shown that either increases or decreases in MPV can indicate various inflammatory statuses and diseases . Interprs-Klotho is significantly associated with well-recognized inflammatory biomarkers. Negative associations were demonstrated with UA, CRP, and WBC count, while a positive association was demonstrated with MPV. UA had the strongest relationship with s-Klotho, suggesting that s-Klotho could serve as an alternative inverse indicator for evaluation of inflammatory states. Further studies are required to assess the applicability of s-Klotho in clinical practice.


In the large-scale breeding of conifers, cultivating embryogenic cells with good proliferative capacity is crucial in the process of somatic embryogenesis. In the same cultural environment, the proliferative capacity of different cell lines is significantly different. To reveal the regulatory mechanism of proliferation in woody plant cell lines with different proliferative potential, we used Korean pine cell lines with high proliferative potential 001#–001 (Fast) and low proliferative potential 001#–010 (Slow) for analysis. A total of 17 glutathione-related differentially expressed genes was identified between F and S cell lines. A total of 893 metabolites was obtained from the two cell lines in the metabolomic studies. A total of nine metabolites related to glutathione was significantly upregulated in the F cell line compared with the S cell line. The combined analyses revealed that intracellular glutathione might be the key positive regulator mediating the difference in proliferative capacity between F and S cell lines. The qRT-PCR assay validated 11 differentially expressed genes related to glutathione metabolism. Exogenous glutathione and its synthase inhibitor L-buthionine-sulfoximine treatment assay demonstrated the positive role of glutathione in the proliferation of Korean pine embryogenic cells. Due to the rapid growth of population and sustained growth of the economy, global demand for wood continues to grow . High-quPinus rigida × P. taeda). Businge et al. [Picea abies. In addition, tryptophan was only presented in two cell lines that can differentiate into somatic embryos. They further inferred that exogenous auxin was crucial to EC proliferation and somatic embryos differentiation. Further research on the regulatory mechanism of EC proliferation in conifers needs to be carried out urgently.Transcriptomics ,14,15,16e et al.  reportedPinus koraiensis Sieb. et Zucc.) is a dominant tree species of temperate zone climax community broad-leaved forest. It is also an extremely important high-quality precious timber tree and nut species and economic forest species in East Asia. The somatic embryogenesis technology system of Korean pine has been established previously [Korean pine  and Slow  cell lines was different. The F cell line had abundant filamentous early somatic embryos on the surface a and a rThe F and S cell lines  were sequenced by Illumina Hiseq platform. A total of 40 Gb clean data was obtained. The clean data in each sample were more than 6 Gb, and the base ratio of Q30 was 93.08–95.93%. The impurities of reads were removed to obtain clean reads. The GC content was more than 43.78%, which met the requirements of subsequent transcriptome de novo assembly. A total of 16,027 unigenes was obtained from de novo assembly, with high assembly integrity. The mapped ratio values of the six samples reached above 75.35%, so the result of de novo assembly in six samples was good and met the requirements for further tests.−5) and HMMER . Principal component analysis (PCA) was performed on six samples to understand the correlation of overall DEGs between the two samples of F and S cell lines and the difference of DEGs between F and S cell lines. According to the PCA diagram, there was little difference in the principal components in the samples of F and S cell lines  between F and S cell lines, in which 870 genes were upregulated and 1013 genes were downregulated . A total of 1422 DEGs with annotation information was obtained by BLAST  metabolism, ascorbate and aldarate metabolism, pyrimidine metabolism, and other pathways  trended toward downregulation, and only a few differential genes (3) trended toward upregulation.A total of 17 GSH-related DEGs was significantly changed between F and S cell lines , includiThrough the observation of transcriptome data, it was found that many DEGs were involved in a plant hormone signal transduction pathway, mainly involved in the metabolic pathway of indole-3-acetic acid (IAA) and cytokine in biosynthesis. Therefore, in this study, genes that played a role in the anabolism, transport, and signal transduction of these two hormones were selected for specific analysis.SAUR family , the expression trends of the remaining 11 DEGs were similar to the RNA-Seq results, indicating that the differential genes obtained by sequencing in this study were credible.Twelve DEGs related to GSH and auxin between F and S cell lines of Korean pine were selected for qRT-PCR verification . Principal component analysis (PCA) was performed on six samples to understand the correlation of overall metabolisms between the two samples of F and S cell lines and the difference of metabolites between F and S cell lines. According to the PCA diagram, there was little difference in the principal components in the samples of F and S cell lines , while tp < 0.05, and VIP > 1). All DEMs were divided into 27 categories, mainly 31 kinds of carboxylic acids and their derivatives; 17 kinds of fatty acyl compounds; 11 kinds of organic oxygen compounds; 6 kinds of imidazopyrimidines; 6 kinds of purine nucleosides and organic compounds; 4 kinds of nitrogen compounds; as well as 3 kinds of benzene and its substitutes, indole and its derivatives, phenols; purine nucleotides; pyrimidine and its derivatives, pyrimidine nucleosides, and pyrimidine nucleotide metabolites; 2 kinds of cinnamaldehyde, cinnamic acid and its derivatives and flavonoid metabolites; and a kind of (5’->5’)-dinucleotide, 5’-deoxyribonucleoside, biotin, and its derivatives.Volcano plot revealed 412 differential metabolites (DEMs) between F and S cell lines, in which 183 metabolites were upregulated and 229 were downregulated  were preferentially analyzed. The KEGG co-enrichment results are shown in The selected genes and metabolic pathways  was performed on the differential genes and metabolites related to GSH for further exploring the gene most related to metabolites . It was The intracellular GSH content of F and S cell lines was shown in p > 0.05). In addition, the intracellular zeatin (ZR) (The results of intracellular hormone content measurement in F and S cell lines are shown in tin (ZR) f and abstin (ZR) g contenttin (ZR) h, ZR/ABAtin (ZR) i, and (Itin (ZR) j were siExogenous addition of GSH promoted EC proliferation in F and S cell lines, and exogenous BSO inhibited EC proliferation k,l, whicThe proliferative process of Korean pine EC was the result of the dynamic balance of embryonic cell division and differentiation. This process was closely related to GSH and plant growth regulators, which was consistent with Belmonte et al. . In the GSH is an important antioxidant in plants, which can protect cells from oxidative stress . It playGST family regulated GSH S-transferases. GST is a family of proteins with various forms and similar three-dimensional structures. GSH S-transferase is a key enzyme in the GSH binding reaction and possesses a well-defined GSH binding domain at its active site, which catalyzed the initial step of GSH binding reaction [GST family, most of which were downregulated, and only one gene was upregulated. Therefore, it was speculated that the stress resistance of the F cell line was higher than that of the S cell line.Genes of the reaction . The comDHAR family. Its expression in the F cell line was higher than that in the S cell line, and its expression was downregulated in the S cell line. This further suggested that the stress resistance of the F cell line might be higher than that of the S cell line, which needed to be further investigated.GSH dehydrogenase/transferase family genes are regulated by dehydroascorbate reductase. Dehydroascorbate reductase (DHAR) is an important antioxidant enzyme in plants. It is the key enzyme to promote ascorbic acid (ASA) regeneration in the process of ASA-GSH cycle, and it plays an important role in protecting cell components against oxidative damage . DHAR caCARP is a leucine peptidase family gene. The overexpression of CARP increased the catalytic activity of several aminopeptidase substrates [CARP family gene in the S cell line trended toward downregulation. In the GSH metabolic pathway, γ-glutamylcysteine, glycine (Gly), and ATP were catalyzed by GSH synthase (GSS) to form GSH, ADP, and P1 [bstrates . CARP acbstrates . In this, and P1 . TherefoAUX1 family genes are auxin influx carriers that mediate the transport of amino-acid signaling molecules. AUX1 family gene restored auxin reactivity and promoted growth [TIR1 family proteins, and binding of auxin to TIR1 can stabilize the interaction between TIR1 and Aux/IAA, thus leading to ubiquitination of Aux/IAA and binding to DNA for regulating auxin-related gene expression [GH3 is an early gene regulated by auxin [GH3 family genes catalyzed free IAA into IAA amino acids and regulated the level of endogenous auxin by regulating the conversion of free and conjugated auxin.An auxin signal transduction pathway between F and S cell lines in Korean pine is shown in d growth . Auxin spression . GH3 is by auxin . GH3 famARF) family genes are auxin response factors. A high concentration of endogenous IAA is conducive to binding with receptors and releasing ARF [ARF gene in the S cell line trended toward downregulation, indicating that the receptor role of TIR1 in auxin signal transduction might be weakened, and the gene expression mode of the F cell line was conducive to the accumulation, transportation, and signal transmission of IAA level. Therefore, it was considered that the level of IAA in the F cell line might be higher than that in the S cell line, and IAA was involved in the regulation of EC proliferative ability in Korean pine. However, there was no significant difference in the expression of auxin-related metabolites between F and S cell lines. By measuring the intracellular hormone content, there was no significant difference in the contents of IAA, ZR, and ABA in F and S cell lines, but IAA/ABA, ZR/ABA and (IAA + ZR)/ABA were significant differences. Therefore, it can be seen that the proportion of growth-promoting hormones in the F cell line was higher than that in the S cell line. The proliferation of Korean pine EC was controlled by the synergistic effect of various hormones in cells.The plant hormone auxin modulates cell proliferation and cell expansion in part by changing gene expression. Primary auxin response genes consist of members of three gene families: Aux/IAA, GH3, and SAUR . The IAAsing ARF . The expFull sibling family cones 1# were collected from a Korean pine seed orchard of Lushuihe Forestry Bureau of Heilongjiang Province 1# on 1 July 2018. The cones were washed with 50-fold dilution detergent for 30 min, rinsed continuously with running water for 8 h, and then placed in an ultra-clean workbench for sterilization with 75% alcohol for 50 min. Then, they were taken out, soaked in 75% alcohol for 1 min, and washed 3–5 times with sterile distilled water.−1), sucrose (30 g·L−1), acid hydrolyzed casein (500 mg·L−1), 1-Naphthylacetic acid (2 mg·L−1), 6-benzylaminopurine (1.5 mg·L−1), and gelrite (4 g·L−1) . Five explants were inoculated into each cultural dish . After adjusting pH to 5.8, we carried out high-temperature and high-pressure sterilization , cooled the cultural medium to 55–60 °C, and added L-glutamine by filtration sterilization (0.22 μm filter). After inoculating, the cultural dishes with explant were cultured in the dark at 23 ± 2 °C.Korean pine megametophyte was used as an explant and put in EC induction medium. The EC induction medium was mLV  basic me−1), 2,4-dichlorophenoxyacetic acid (2.26 μmol·L−1), 6-benzylaminopurine (0.44 μmol·L−1), acid hydrolyzed casein (500 mg·L−1), L-glutamine (500 mg·L−1), and gelrite (4 g·L−1). The EC was sub-cultured every two weeks. The initial inoculation amount of EC was 0.2 g, 3 replicates per cell line, and the cultural conditions were the same as those of induction culture.When the EC cluster grew to a diameter of 1–2 cm, it was transferred to the proliferation medium. The medium was mLV basic medium supplemented with sucrose  with high proliferative potential and 001#–010 , with low proliferative potential. The cell lines F and S were used for cytological observation. The method used was as follows: fresh EC was put on the slide, stained with 0.1% safranine dye for 10 min, and covered with a cover glass. The cover glass was gently tapped to spread the plant tissues evenly. Then, it was immediately observed and recorded under an optical microscope .To explore the mechanism that accounted for the difference in proliferative potential of different embryogenic cell lines of Korean pine, we used two EC cell lines F and S as tested materials, performed transcriptomic and metabolomic analyses, and subsequently identified multiple key genes and metabolites regulating EC proliferative potential. After 14 days of culture, EC was collected and quickly frozen in liquid nitrogen and stored in −80 °C refrigerator for transcriptome sequencing analysis. The samples were taken out from the −80 °C freezer and used to extract RNA with the RNA Extraction Kit . Then, 1 μL of the RNA solution was used to detect the concentration using Nanodrop. RNA integrity was assessed using the Agilent Bioanalyzer (2100 RNA Nano 6000).A total of 1 μg of RNA was used for the construction of sequencing libraries with different index barcodes indicating different samples. Finally, the library fragments were purified using the AMPure XP system and the library quality was assessed on the Agilent Bioanalyzer 2100 system. Indexed/barcoded samples were clustered on the cBot cluster generation system using the TruSeq PE Cluster Kit v3-cBot-HS (Illumia). After cluster generation, library preparations were sequenced on the Illumina Hiseq platform and paired-end reads were generated. The experiments were performed in triplicate.The test data were collated by Excel 2003, and BLAST  software−ΔΔCt calculation method, and the calculation was performed according to the method of Livak [The AAE7  gene of Korean pine was used as the internal control gene and twelve DEGs were validated by qRT-PCR. The primers were synthesized by Beijing biomarker technologies corporation. The details of the primer sequences are shown in of Livak , with 3 ECs grown from the F and S cell lines were placed in a lyophilizer (Scientz-100F) for vacuum freeze-drying. After being fully dried, they were thoroughly ground into powder  with a grinder . A total of 100 mg of powder was dissolved in 1.2 mL of 70% methanol solution for extraction . The extracted mixture centrifuged at 12,000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter for UPLC-MS/MS analysis . The experiments were performed in triplicate.p < 0.05, and VIP > 1 and the screening criteria for differential expression of genes with fold difference ≥ 2, FDR ≤ 0.01.The above transcriptome and metabolite identification results were analyzed jointly. Set the criteria: the screening criteria for differential expression of metabolites with folded difference (FC) > 1, The content of GSH was determined by a Nanjing Jiancheng kit . The model of glutathione oxidized (GSSG) content kit was purchased from Nanjing Jiancheng Bioengineering Institute, A 061-1. The calculation method of total glutathione (T-GSH) content was the sum of GSH and GSSG. GSH: GSSG was described as the ratio of GSH to GSSG.The contents of indole-3-acetic acid (IAA), zeatin (ZR) and abscisic acid (ABA) in the F and S cell lines were determined by an enzyme-linked immunosorbent assay (ELISA) kit , and the specific assay method was shown in the kit instruction.−1 GSH  or 0.5 mmol·L−1 BSO , the treatment without GSH and BSO was used as the control (CK), the fresh weight was measured on the 14th day of EC proliferation culture, and the EC proliferative rate was calculated.F and S cell lines EC (0.2 g) were inoculated into the mLV basic medium supplemented with 0.5 mmol·Ln = 5. Graphs were constructed in Sigma Plot 12.0 .Experimental calculations, including proliferation parameters, were performed using Excel 2003 . A one-way analysis of variance (ANOVA) and Duncan’s multiple comparisons tests were performed using SPSS 19 , mean ± se, There were differences in proliferative potential among different embryogenic cell lines of Korean pine. Integrated transcriptome–metabolome analysis shows that glutathione-related genes and metabolites play a positive role in the proliferation of embryogenic callus of Korean pine. Through the analysis results, it was found that the glutathione content in the F (Fast proliferative rate) cell line was significantly higher than that in the S (Slow proliferative rate) cell line. When exogenous glutathione was added to the two cell lines, the proliferation rate of both cell lines was promoted, which further confirmed the results of transcriptome and metabolome. Our research laid a foundation for revealing the regulatory mechanism of woody plant cell proliferation and cell totipotency expression, established the technology to improve woody plant embryogenic callus proliferative ability, optimized the woody plant somatic embryogenesis system, and provided a new ground for revealing the molecular regulatory mechanism of conifer somatic embryogenesis.


Deregulation of tight junction (TJ) proteins and the associated disruption of TJ function has been demonstrated to play a role in the development of endometrial cancer. In the current study, we have shown overexpression of claudin-3 and -4 mRNA (by RT-PCR) and protein (by immunoblotting) in a panel of 9 human endometrial cancer cell lines. To further expand our understanding of the complex role of TJ deregulation in endometrial cancer, we also investigated the expression of 84 TJ and TJ-associated genes  by microarray analysis. Consistent with the claudin-3 and -4 RT-PCR and immunoblot findings described above, we observed overexpression of the claudin-3 and -4 genes by microarray analysis. Further, we observed overexpression of an additional three genes in 8 of the 9 endometrial cancer cell lines: OCLN (occludin), F11R (JAM-A) and TJP3 (ZO-3). OCLN and F11R encode integral membrane proteins whereas TJP3 encodes a cytosolic scaffolding protein that indirectly links membrane TJ proteins to the actin cytoskeleton and cell signaling pathways. Our data suggest that the structural disruption of TJs coupled with the downstream deregulation of signaling pathways involved in cellular proliferation and migration may contribute to the development of endometrial cancer. Endometrial cancer is the most common female reproductive cancer in the United States and so far has accounted for an estimated 12,550 deaths in 2022 Over the last few years, we and others have demonstrated a role for tight junction (TJ) deregulation in the development of endometrial cancer The TJ protein complex consists of the integral membrane proteins: claudins (CLDNs), occludin (OCLN), junctional adhesion molecules (JAMs) and the coxsackievirus and adenovirus receptor (CAR). Investigations into the role of TJ deregulation in endometrial cancer have primarily focused on the expression of CLDNs and OCLN. Indeed, several studies have shown overexpression of CLDNs-3 and -4 in uterine serous papillary carcinoma In the current study we have demonstrated the deregulated expression of CLDNs-3 and -4 in a panel of human endometrial cancer cell lines. In addition, to gain a more global understanding of the role of TJ deregulation in endometrial cancer development we have evaluated those same cell lines for the expression of 84 TJ and TJ-associated genes through microarray analysis.Our study found that all but one of the cell lines showed overexpression of an additional three TJ genes: OCLN, F11R (JAM-A) and TJP3 (ZO-3), suggesting a more complex role for TJ deregulation in endometrial cancer involving proteins that function at different levels within the TJ network from cell to cell adhesion to cell signaling.2 humidified atmosphere at 37oC.The normal endometrial cell line THESCs (CRL-4003) and the endometrial cancer cell lines, AN3-CA (HTB-111), KLE (CRL-1622), MES-SA (CRL-1976), RL95-2 (CRL-1671), and SK-UT-1 (HTB-114) were obtained from the American Type Culture Collection . The endometrial cancer cell lines EFE-184 (ACC-230), MFE-280 (ACC-410), MFE-296 (ACC 419) and MFE-319 (ACC 423) were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen DSMZ . The cancer cell lines were cultured in their respective medium as stipulated by ATCC or DSMZ supplemented with 10-20% fetal bovine serum (FBS) , 1% penicillin/streptomycin/2mM-glutamine . The normal endometrial cell line (THESCs) was cultured in phenol-free medium per ATCC instructions supplemented with 1.5 g/l sodium bicarbonate, 1% ITS+ Premix , 500ng/ml puromycin, 10% charcoal/dextran treated fetal bovine serum (CSFBS) . All cell lines were maintained in a 5% COoC for 3 min, sheared and stored at -80°C.Cells were harvested by washing with PBS, followed by treatment with trypsin/EDTA (0.25%/1mM). Protein extracts were obtained by resuspending the cell pellet in Laemmli sample buffer/5% β-mercaptoethanol . The extracts were boiled at 1006 cells/ml) using the Qiagen RNeasy Mini Kit  according to the manufacturer's instructions. RNA extracts were stored at -80°C.RNA was isolated from endometrial cells  according to the manufacturer's instructions, then the signals were visualized using the Invitrogen™ iBright™ FL1500 . As a loading control, membranes were re-probed with 4.0µg/ml mouse monoclonal anti-human GAPDH .Approximately 30 µg of each of the protein extracts was subjected to electrophoresis on precast 10% SDS-polyacrylamide gels  and electrophoretically transferred to Immobilon-P PVDF membranes . The membranes were probed for 1 h at room temperature with 2 μg/ml rabbit polyclonal anti-human claudin-3  or 3 μg/ml mouse monoclonal anti-human claudin-4  primary antibodies in 5% milk/PBS solution. The membranes were washed in PBS for 10 min, PBS/0.1% TWEEN for 5 min (twice) then PBS for 10 min. The membranes were then incubated with 1:3000 HRP-conjugated goat anti-rabbit  or goat anti-mouse  secondary antibodies for 1 h at room temperature then washed as described above. Membranes were incubated with the enhanced chemiluminescence ECL® RT-PCR kit according to the manufacturer's instructions . Briefly, a 20 μl reaction contained 2 μg of RNA, 1X Buffer RT, 5 mM dNTPs, 10 U/ml of RNase inhibitor, and 1 U/ml of reverse transcriptase. The SYBR Green I assay and Applied Biosystems StepOnePlusTM were used to detect RT-PCR products . Each 20 μl RT- PCR reaction contained 0.1 μg of cDNA; 400 nM each of the following primer sequences taken from Pan et al. . The melting curve was measured at 65-95oC with a heating rate of 0.1oC per sec and a continuous fluorescence measurement. Each reaction was performed in triplicate. The relative expression was calculated using the 2 -△△Ct method . Statistical significance was evaluated for each target gene separately using one-way ANOVA and post-hoc Tukey HSD test compared to THESCs (n = 4); *p<0.05; ** p<0.01 was considered to indicate a statistically significant result.Prior to reverse transcription, RNA samples were treated with DNase I . RNA was then reversed transcribed into cDNA using the Q-OMNISCRIPT2 Profiler PCR Array  and the Applied Biosystems StepOnePlusTM (Applied Biosystems), the expression of 84 tight junction genes was determined. In addition to the tight junction genes, the 96-well RT2 profiler microplate contained primers for 5 housekeeping genes, one DNA contamination control, three reverse transcription controls and three positive PCR controls. Briefly, the RNA from endometrial cancer cell lines and the normal endometrial cell line (THESCs) was isolated using the RNeasy Mini Kit  according to the manufacturer's instructions. RNA samples were reverse transcribed into cDNA using the RT2 first strand kit . cDNA was mixed with the RT2 SYBR Green mastermix  and 25 μl of the PCR mix was aliquoted into each well of the RT2 profiler PCR array microplate. The PCR program consisted of an initial denaturation period (10 min at 95oC) followed by 40 cycles of amplification and quantification . The relative expression was based on the expression ratio of each of the TJ genes and was determined using the 2 -△△Ct method as described above .Using a SYBR green-based quantitative real-time PCR Human Tight Junctions RTThe expression of CLDN-3 and -4 proteins in a panel of nine endometrial cancer cell lines was determined by immunoblot analysis Figure B and 2B.To investigate the mechanism of CLDN-3 and -4 protein overexpression, we subjected RNA isolated from the same panel of endometrial cancer cell lines to RT-PCR analysis using primers targeted to the CLDN-3 and -4 genes Figure A and 2A.2 Profiler PCR microarray  using the RTay Table . As expeay Table . In addiIn the current study, we investigated the contribution of deregulated TJ gene expression to endometrial cancer development. Using immunoblot analysis, we demonstrated overexpression of the CLDN-3 and -4 integral membrane proteins in 4 of 9 and 5 of 9 endometrial cancer cell lines, respectively. RT-PCR analysis of the same cell lines indicated increased mRNA transcription as the underlying mechanism for the protein overexpression. In the case of two cell lines, there were inconsistencies between the patterns of CLDN protein and mRNA expression. AN3-CA, showed overexpression of the CLDN-3 protein but no associated overexpression of the CLDN-3 mRNA. This finding could be due to stabilization of the CLDN-3 protein in this cell line. In contrast, the MFE-280 cell line showed overexpression of CLDN-4 mRNA but no associated protein overexpression. It is possible that defective RNA processing may be responsible for this discrepancy.Consistent with our findings, multiple groups have shown deregulated expression  of CLDN-3 and -4 integral membrane proteins To-date, much research has focused on the role of deregulated claudin and occludin proteins in cancer development. However, these integral membrane proteins constitute just one component of the complex network of TJ proteins that contribute to a wide and diverse array of cellular processes. To gain a greater understanding of the role of TJ deregulation in cancer development, we expanded our analysis beyond integral membrane proteins to investigate the expression of genes encoding TJ-associated proteins found in the cytosol and cytoskeleton. Using a TJ microarray, we were able to analyze the expression of 84 different TJ genes. In addition to the expected frequent overexpression of the much studied CLDN-3, CLDN-4 and OCLN genes, we also observed overexpression of an additional 21 TJ genes in at least one of the endometrial cancer cell lines. Notably, a further two genes - F11R (JAM-A) and TJP3 (ZO-3) were overexpressed in 8 of the 9 cell lines. These findings are consistent with a study by Colas et al. (2011) By studying the expression of all of the known TJ and TJ-associated genes in a panel of endometrial cancer cell lines, our microarray analysis indicates a more complex role for TJ deregulation in tumor etiology. Specifically, we have confirmed the role of deregulated CLDN and OCLN genes  and provided strong additional evidence for deregulated F11R (JAM-A) and TJP3 (ZO-3) in the development of endometrial cancer. These data provide potential gene candidates for the development both of diagnostic markers and novel gene-based therapeutic approaches to the treatment of endometrial cancer.


Cutaneous T-cell lymphoma (CTCL) is a severe lymphoid malignancy with a worse prognosis lacking curative treatment regimens. Several gene mutations and deregulated pathways, including NFkB signaling, have been implicated in its pathogenesis. Accordingly, CTCL cell line HUT-78 reportedly contains mutated NFKB2, which is constitutively activated via partial gene deletion, also demonstrating that genomic rearrangements cause driving mutations in this malignancy. Here, along with HUT-78, we analyzed CTCL cell line HH to identify additional aberrations underlying gene deregulation. Karyotyping and genomic profiling of HH showed several rearrangements worthy of detailed investigation. Corresponding to the established karyotype, RNA-seq data and PCR analysis confirmed the presence of t, generating a novel fusion gene, FOXK2::TP63. Furthermore, chromosomal rearrangement t was connected to amplification at 4q24–26, affecting aberrant NFKB1 overexpression thereat. Transcription factor binding-site analysis and knockdown experiments demonstrated that IRF4 contributed to NFKB1 expression. Within the same amplicon, we identified amplification and overexpression of NFkB signaling activator CAMK2D (4q26) and p53-inhibitor UBE2D3 (4q24). Genomic profiling data for HUT-78 detailed a deletion at 10q25 underlying reported NFKB2 activation. Moreover, amplifications of ID1 (20q11) and IKZF2 (2q34) in this cell line drove overexpression of these NK cell differentiation factors and possibly thus formed corresponding lineage characteristics. Target gene analysis for NFKB1 via siRNA-mediated knockdown in HH revealed activation of TP63, MIR155, and NOTCH pathway component RBPJ. Finally, treatment of HH with NFkB inhibitor demonstrated a role for NFkB in supporting proliferation, while usage of inhibitor DAPT showed significant survival effects via the NOTCH pathway. Collectively, our data suggest that NFkB and/or NOTCH inhibitors may represent reasonable treatment options for subsets of CTCL patients. Cutaneous T-cell lymphoma (CTCL) covers a heterogeneous group of lymphomas presenting in the skin. This malignancy becomes manifest in subtypes with distinct histopathologies and clinical features and includes mycosis fungoides, Sézary syndrome, lymphomatoid papulosis, and primary cutaneous anaplastic large cell lymphoma . CTCL isAnalyses of chromosomal and genomic aberrations in CTCL patients and cell lines have shown the presence of translocations, fusion genes, and copy number alterations ,5,6,7,8.Deregulated signaling pathways in CTCL patients include the JAK-STAT pathway, MAP kinase signaling, p53 pathway, and NFkB signaling . The NFkChromosomal aberrations and copy number alterations underlying activation of oncogenes or inhibition of tumor suppressor genes have been frequently described in CTCL ,4,5,6,7.Cell lines HH, JURKAT, NK-92, KHYG-1, and DERL-2 were obtained from the DSMZ , HUT-78 from the ATCC , and the cell lines MAC-1 and MAC-2A kindly provided by Marshall E. Kadin . Cell culture conditions and backgrounds of each cell line are described elsewhere . Cell li6 cells by electroporation using the EPI-2500 impulse generator  at 350 V for 10 ms. Transfected cells were harvested after 20 h cultivation. Transfection experiments were performed twice, generating similar results.Gene-specific siRNA oligonucleotides and AllStars negative control siRNA (siCTR) were purchased from Qiagen . SiRNAs (80 pmol) were transfected into 1 × 10Additional cell treatments were performed with 14 µM NFKB inhibitor  or with 10 µM DAPT -L-alanyl]-S-phenylglycin-tert-butylester; Sigma), both dissolved in dimethyl sulfoxide (DMSO). Treated cells were functionally analyzed with the IncuCyte S3 Live-Cell Analysis System . This system allows continuous documentation of living cells within the incubator. Subsequent analysis of the microscopical images using the associated software allows adapted examination of the data, graphical presentation, and statistical calculations. For detection of apoptotic cells, we additionally used the IncuCyte Caspase-3/7 Green Apoptosis Assay diluted at 1:2000 . This assay stains apoptotic cells detectable by integrated fluorescence microscopy. Thus, proliferating and apoptotic cells are detectable at once. Live-cell imaging experiments were performed twice with four-fold parallel tests.Karyotyping, spectral karyotyping (SKY), and fluorescence in situ hybridization (FISH) were performed as described previously . BAC cloFor genomic profiling, the genomic DNA of CTCL cell lines was prepared by the Qiagen Gentra Puregene Kit (Qiagen). Labeling, hybridizatin, and scanning of HD Cytoscan arrays were performed at the Genome Analytics Facility of the Helmholtz Centre for Infection Research , using HD arrays according to the manufacturer´s protocols . Data were visualized and interpreted using the Chromosome Analysis Suite software version 2.0.1.2 (Affymetrix).www.ebi.ac.uk/ena; accessed on 1 March 2022) using data set E-MTAB-7721 [https://github.com/pinin4fjords/shinyngs; accessed on 1 March 2022) for bar plots and Heatmapper for heatmaps [www.proteinatlas.org; accessed on 1 March 2022) [Transcriptome data from 100 leukemia/lymphoma cell lines (LL-100) were obtained from the European Nucleotide Archive  .https://www.ncbi.nlm.nih.gov/gds/; accessed on 1 March 2022) as reported previously [Expression profiling data sets from cell lines were generated by Dr. Robert Geffers  using HG U133 Plus 2.0 gene chips  and obtained from GEO . cDNA was synthesized by random priming from 5 µg RNA using Superscript II (Invitrogen).https://blast.ncbi.nlm.nih.gov/Blast.cgi; accessed on 2 March 2022).To analyze fusion genes in cell lines, we used the following oligonucleotides: FOXK2-for 5′-GGTCTTCAGGGTACAAGGTGG-3′, FOXK2-rev 5′-GCCCTTGTCCGCAGTCCTGTAG-3′, TP63-for1 5′-TGAACCATCAGAAGATGGTGCG-3′, TP63-rev1 5′-GCATCGAAGGTGGAGCTGGGC-3′, TBL1XR1-for 5′-GAAAGCCATATCAGTCAGTCC-3′, TBL1XR1-rev 5′-TCTATCAGGGACAGAGACTCT-3′, TP63-for2 5′-GTTCAGCCCATTGACTTGAAC-3′, TP63-rev2 5′-CTGCTGGTCCATGCTGTTC-3′. To analyze a described mutation in NFKB1, we amplified the according gene fragment using cDNA as a template and oligonucleotides NFKB1-for 5′-CTTCAGAATGGCAGAAGATGATCC-3′ and NFKB1-rev 5′-CAGGTAGTCCACCATGGGATG-3′. PCR products were generated using taqpol (Qiagen) and thermocycler TGradient , analyzed by gel electrophoresis and documented with the Azure c200 Gel Imaging System . Oligonucleotides were obtained from Eurofins MWG . PCR products were cloned into pCR4-TOPO (Invitrogene) and sequenced at Eurofins MWG. The sequences were analyzed using nucleotide BLAST  PCR analysis was performed with the 7500 Real-time System, using commercial buffer and primer sets . For normalization of expression levels, we analyzed the transcript of TATA box binding protein (TBP). We used the ddCT method, and the obtained values are indicated as fold expressions in relation to one selected sample that was set to 1. For quantification of genomic copy numbers for ID1, we used the following oligonucleotides: ID1-1 5′-CTACGACATGAACGGCTGTTACTC-3′ and ID1-2 5′-TTCGGATTCCGAGTTCAGCTCC-3′. The MEF2C locus was used as a control: MEF2C-1 5′-AGAAGGCTTATGAGCTGAGC-3′ and MEF2C-2 5′-AGACTGGCATCTCGAAGTTG-3′. Genomic DNA was prepared as described above.t-test and derived p-values indicated by asterisks .Quantitative PCR analyses were performed in triplicate. Standard deviations are calculated for each experiment and presented in the figures as error bars. Statistical significance was assessed by Western blots were generated by the semi-dry method, and protein lysates from cell lines were prepared using SIGMAFast protease inhibitor cocktail (Sigma). Proteins were transferred onto nitrocellulose membranes  and blocked with 5% dry milk powder dissolved in phosphate-buffered saline buffer (PBS). The following antibodies were used: alpha-Tubulin (Sigma), NFKB1 , and NFKB2 . The used antibodies were diluted as follows: alpha-Tubulin (TUBA) 1:1000, NFKB1 1:1000, NFKB2 1:500. For loading controls, blots were reversibly stained with Poinceau (Sigma), and detection of TUBA was performed thereafter. Secondary antibodies were linked to peroxidase for detection by Western-Lightning-ECL . Western blot analyses were performed twice. Documentation was performed using the digital system ChemoStar Imager .Chromosomal and genomic aberrations may result in the deregulation of specific genes contributing to the pathogenesis of hematopoietic malignancies. To identify novel oncogenes that may potentially serve as therapeutic targets in CTCL, we used cell line HH as a model and searched for chromosomal aberrations by performing classical karyotyping, SKY, and FISH analyses A. The coFusion genes form a special class of oncogenes with proven pathological and diagnostic potential. To check if cytogenetic aberrations in cell line HH generated fusion genes, we analyzed our published LL-100 RNA-seq data set bioinformatically. This procedure revealed a potential fusion between FOXK2 at 17q25 and TP63 at 3q28, highlighted by the cytogenetic data showing explicit der(17) partners of the semi-cryptic t. According to the RNA-seq data and verified by RT-PCR and Sanger sequencing analyses, we observed the expression of a FOXK2::TP63 fusion transcript in HH, joining FOXK2 exon 4 and TP63 exon 4 B. In addInterestingly, T-cell large granular lymphocyte leukemia (T-LGL) cell line MOTN-1 also showed elevated TP63 levels . AccordiWe also performed genomic profiling of CTCL cell lines HH and HUT-78, revealing copy number alterations that may underlie specific gene deregulations. The results for all chromosomes are shown in To investigate transcriptional consequences of the genomic amplification at 4q24–26, we analyzed LL-100 RNA-seq data of HH in comparison to 11 leukemia/lymphoma control cell lines comprising DERL-7, MOTN-1 (T-cell lymphoma), CCRF-CEM, JURKAT, RPMI-8402, MOLT-4, HPB-ALL, DND-41 (T-cell leukemia), and YT, NK-92, KHYG-1 (NK-cell leukemia/lymphoma). We assigned 49 amplified genes and illustrated their activities in a heatmap A. For HHExpression data for NFKB1 using the complete LL-100 RNA-seq data set and RQ-PCR analysis of NFKB1 in CTCL and T-ALL cell lines showed high transcript levels exclusive to HH B,C. ReceFinally, Western blot analysis of NFKB1 and NFKB2 in CTCL and control cell lines showed results corresponding to the RNA data. Accordingly, NFKB1 was highly expressed in HH, while NFKB2 was elevated in all analyzed CTCL cell lines, including HH, HUT-78, MAC-1, and MAC-2A C. Taken www.genome.cse.ucsc.edu/index.html; accessed on 1 March 2022). This exercise revealed several IRF sites . Fusion genes containing TP63 have been reported in various T-cell malignancies, including CTCL ,12. ThesAdditionally, we detected the fusion gene TBLXR1::TP63 in cell line MOTN-1. Both FOXK2 and TBLXR1 are ubiquitously expressed in hematopoietic cell lines. In contrast, TP63 is expressed in selected cell lines at various levels. Thus, wild-type-configured TP63 is expressed in HH but not in MOTN-1. However, in both cell lines, the level of TP63 transcript was enhanced, possibly due to the fusion to FOXK2 or TBLXR1, respectively.Chromosomal rearrangement t in HH was intimately connected with the complex amplification at 4q24–26, targeting the NFKB1 locus. NFKB1 and NFKB2 are functionally related and encode TFs, which execute gene regulation via the NFkB signaling pathway. This pathway plays an oncogenic role in several hematopoietic malignancies, including CTCL ,49. Our Target gene analysis of NFKB1 revealed transcriptional activation of MIR155 and RBPJ. MIR155 encodes the micro-RNA miR155, which affects the suppression of specific genes and operates as an oncogene in several hematopoietic malignancies, including CTCL ,34. The RBPJ encodes a TF that interacts with the activated intracellular NOTCH receptor to regulate NOTCH target genes. The aberrantly activated NOTCH pathway plays an oncogenic role in T-cell malignancies, including CTCL ,36,37. OThe amplicon at 4q24–26 also contained the genes CAMK2D and UBE2D3, which are involved in NFkB signaling and p53 degradation, respectively. CAMK2D encodes a protein kinase that binds and inhibits the repressor-protein IKKB, resulting in activation of NFkB signaling . DownregFinally, we identified enhanced expression of ID1 and IKZF2 in CTCL cell line HUT-78. These TFs are involved in NK-cell differentiation and may, thus, shift the phenotype of HUT-78 toward NK-cells as indicated by cluster analysis ,41. CTCLWe have identified widespread genomic alterations in CTCL cell line HH, separately generating the fusion gene FOXK2::TP63, overexpression of NFKB1, and three different modes of p53 suppression. Our data highlight the pathogenic impact of NFkB and NOTCH signaling in CTCL, recommending therapeutic targeting of these pathways. CTCL cell lines HH and HUT-78 differ in mode and type of aberrantly activated NFkB factors and may, thus, serve to develop adjusted treatment protocols for affected patient subsets. However, additional analyses are required to support our findings in patients.


H-3,6-diazaphenothiazine (DPT-1) and pentacyclic 7-(3′-dimethylaminopropyl)diquinothiazine (DPT-2), have recently been shown to exhibit promising anticancer activities in vitro. In this report, we demonstrated that DPT-1 and DPT-2 could be pro-apoptotic agents in lung carcinoma, the human lung carcinoma A549 and non-small lung carcinoma H1299, in the range of IC50 = 1.52–12.89 µM, with a protective potential to healthy cell lines BEAS-2B and NHDF. The compounds showed higher activity in the range of the tested concentrations and low cytotoxicity in relation to normal healthy cells than doxorubicin, used as the reference drug. The cytostatic potential of DPT-1 and DPT-2 was demonstrated with the use of MTT assay. Cell cycle analysis via flow cytometry using Annexin-V assay showed the pro-apoptotic and pro-necrotic role of the studied diazaphenothiazines in the cell cycle. DPT-1 and DPT-2 initiated a biological response in the investigated cancer models with a different mechanism and at a different rate. Based on these findings, it can be concluded that DPT-1 and DPT-2 have potential as chemotherapeutic agents.The phenothiazine derivatives, tricyclic 10 Worldwide, lung cancer has been the most common diagnosed carcinoma for the last several decades. It is the world’s leading cause of cancer death ,2. This The heterocyclic ring system plays an important role in the development of novel scaffolds with improved pharmaceutical properties in anticancer research . TricyclDPT) series, two derivatives deserve special attention: tricyclic 10H-3,6-diazaphenothiazine   and pentacyclic 7-(3′-dimethylaminopropyl)diquinothiazine   exhibited an extremely strong action against the glioblastoma SNB-19, melanoma C-32, and breast cancer MCF-7 cell lines with IC50 values of 0.46 and 0.72 μg/mL and non-toxic action against the normal fibroblast HFF-1 cell line   ,27,29.5 in 2 mL of completed fresh DMEM-F12 medium, 24 h before drugs addition. An Aria III flow cytometer  was used. The flow cytometry analysis was done on a free Flowing Software 2.5.1 program  [Many drugs work by inhibiting cell proliferation and causing cell cycle blockage. Using flow cytometry, the impact of new compounds on the cell cycle were determined. Cells incubated with compound were also analyzed for cellular death pathways, apoptosis induction, followed by Annexin-V assay. Annexin-V is a protein that binds phosphatidylserine and allows early detection of apoptosis; additionally, iodium propide staining distinguishes it from necrosis. For apoptosis and cell cycle assays the cell cultures were plated in 6-well plates at a confluence of 1 × 10Finland) ,27,29.Microscopic observation and images acquisition with Live Cell Analyzer  was used to directly observe on-plate confluence, density, and cell viability and images acquisitions ,31.t-test, and a p-value < 0.05 is indicated with a star [The results are expressed as means ± SD from three independent experiments. Results were analyzed in MS Excel 2010. Statistical significance was calculated with a h a star ,29,30,31In silico analyses of the molecular descriptor and parameters of Lipinski’s, Ghose’s and Veber’s rules and an ADME profile were carried out using a Swiss internet server SwissADME . PredictH-3,6-diazaphenothiazine  and pentacyclic 7-(3′-dimethylaminopropyl)diquinothiazine , demonstrating significant in vitro anticancer activity against lung cancer cell lines, the human lung carcinoma A549 and non-small lung carcinoma H1299, and protective potential to healthy cell lines, BEAS-2B and NHDF. The reference compound in the conducted studies was doxorubicin. Using a 72 h MTT viability assay (Promega) we observed a strong cytotoxic activity of DPT-1 and DPT-2.In this publication, we presented two synthetic diazaphenothiazines, tricyclic 1050 doses against A549 or H1299 cells confirms promising selectivity against cancer. The analysis of the cell cycle revealed different pathways of the mechanism of anticancer activity induced by the studied diazaphenothiazines. DPT-1 activated the process of apoptosis, in contrast to DPT-2, which activated the necrosis phase. Both tested compounds showed selectivity of action. Preliminary in silico analyses of ADME parameters and the biological profile confirmed the validity of our research.Both compounds appear to be good candidates for in vitro tests, especially in 3D cultures, where a mixture of sensitive healthy cell lines is used together with chemoresistant cancer cell lines. A low lethal effect observed in NHDF or BEAS-2B cells at ICDPT-1 and DPT-2 possess potential as chemotherapeutic agents. Further advanced in vivo and enzymatic studies are planned.Based on these primary findings, it can be concluded that