Total knee arthroplasty (TKA) is the most common and cost-effective treatment for older adults with long-standing osteoarthritis. During TKA, muscle cells suffer from prolonged oxygen deficiency, which leads to altered cell metabolism that reduces the energy demand and maintains cell homeostasis before blood flow is restored. This study focused on the role of the lncRNA muscleblind-like 1 antisense RNA 1 (MBNL1-AS1) in protecting sevoflurane-pretreated mice against ischemia-reperfusion (I/R) injury after TKA, as well as the elucidation of the potential associated mechanism. Identification of differentially expressed lncRNAs was performed using the microarray dataset GSE21164, which was extracted from the GEO database. Target genes of the lncRNA were determined using Multi-Experiment Matrix (MEM), a dual-luciferase reporter gene assay, and KEGG enrichment analyses. The results showed that MBNL1-AS1 was overexpressed in skeletal muscle cells in mice, while KCNMA1, which was enriched in the cGMP-PKG signaling pathway, was negatively regulated by MBNL1-AS1. Furthermore, I/R mice displayed serious inflammatory reactions. Down-regulation of MBNL1-AS1 increased the expression of KCNMA1, PKGII, VASP, VEGF, Bcl-2, Cyclin D1, Cyclin D3, and Cdc 42 but decreased the expression of Bax, cleaved caspase-3, and cleaved PARP. Furthermore, upon MBNL1-AS1 upregulation, the rate of cell apoptosis increased while the rate of cell proliferation decreased. Our data suggested that down-regulated lncRNA MBNL1-AS1 might promote the proliferation and inhibit the apoptosis of skeletal muscle cells by upregulating KCNMA1 expression via activation of the cGMP-PKG signaling pathway, thus protecting sevoflurane-pretreated mice against I/R injury after TKA. A potential therapeutic target identified by researchers in China could help limit damage to tissues following osteoarthritic knee surgery. A total knee arthroplasty can alleviate symptoms of end-stage osteoarthritis, but the surgery requires use of a tourniquet. This temporarily cuts blood supply to tissues and can trigger severe ischemia-reperfusion (I/R) injury, tissue damage caused by blood flow returning after oxygen deficiency. Shao-Nan Yu and co-workers at the China-Japan Union Hospital of Jilin University, Changchun, demonstrated that lowering expression of a particular RNA molecule following surgery could limit I/R damage. They found that the molecule was over-expressed in mice during I/R injury. This overexpression limited activation of a signalling pathway and an associated protein vital to the chemical balance of cell membranes and healthy muscle cell contraction. TKA has been widely used in China due to its excellent mid-term prognosis2. During orthopedic procedures such as TKA, tourniquets are generally applied to control blood loss and to maintain a relatively clear surgical field3. However, when tourniquets are used, skeletal muscles can suffer from ischemia, while subsequent reperfusion into ischemic muscles can cause severe complications4. Most importantly, ischemia-reperfusion (I/R) injury caused by the application of tourniquets can lead to oxidative muscle injury and muscle atrophy, which can hinder full functional recovery after TKA surgery. Nevertheless, how I/R injury affects the functions of human skeletal muscles at the cellular level remains poorly understood5. Furthermore, increasing evidence has demonstrated the beneficial role of sevoflurane (Sevo) against I/R injury7. In this study, we aimed to explore the molecular mechanisms underlying the protective role of Sevo against I/R injury after TKA.Total knee arthroplasty (TKA) is one of the most common orthopedic operations in elderly patients with end-stage osteoarthritis (OA), a degenerative disease greatly affecting the quality of life of older adults8. A previous study reported the identification of differentially expressed lncRNAs using the GSE21164 microarray dataset from the GEO database9. In the present study, muscleblind-like 1 antisense RNA 1 (MBNL1-AS1) was determined to be a differentially expressed lncRNA implicated in I/R injury after TKA. Based on data obtained from the Genotype-Tissue Expression (GTEx) project, a database on the variation of gene expression in human tissues10, a correlation  was found between MBNL1-AS1 and potassium calcium-activated channel subfamily M alpha 1 (KCNMA1). Interestingly, in the present study, MBNL1-AS1 was also identified as a putative target gene of KCNMA1 based on the Multi-Experiment Matrix , a web resource of gene co-expression data arranged by different species and types of microarray platforms11. Furthermore, previous Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses based on the WebGestalt database indicated that KCNMA1 participates in the cyclic guanosine monophosphate-protein kinase G (cGMP-PKG) signaling pathway, which plays critical roles in cellular functions14. Therefore, this study aimed to explore the role of MBNL1-AS1 in protecting the skeletal muscle cells of Sevo-pretreated mice against I/R injury after TKA and the role of KCNMA1 and the cGMP-PKG signaling pathway in the underlying molecular mechanism of MBNL1-AS1.Gene Expression Omnibus (GEO) is a public repository database for high-throughput gene sequencing, hybridization arrays, chips, and microarraysThe experimental protocol and animal use plan in this study were approved by the Animal Ethics Committee of China-Japan Union Hospital of Jilin University.http://www.ncbi.nlm.nih.gov/geo) were analyzed using the Affymetrix Human Genome U133 Plus 2.0 Array . Background correction and normalization of GSE21164 were performed with the Affy installation package in R software15. Identification of differentially expressed lncRNAs in GSE21164 was performed using an empirical Bayes method in conjunction with a linear model and t-test16. Putative target genes of lncRNA were determined based on MEM (http://biit.cs.ut.ee/mem/). The target genes enriched in the cGMP-PKG pathway were ascertained using the KEGG pathway database and the WebGestalt database 17.The GSE21164 microarray dataset and probe files for TKA downloaded from the GEO database  were obtained from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences . All mice were kept under specific temperature (18–22 °C), relative humidity (40–70%), and noise (<50 dB) conditions, with ad libitum access to water and food in an animal room with a 12-h light/dark cycle. The mice were randomly assigned to a normal group, an I/R group and a Sevo group (I/R mice treated with Sevo after TKA), with 20 mice in each group.During model establishment, the mice were fixed on a super-clean table, anesthetized by intraperitoneal injection of pentobarbital (30 mg/kg) and disinfected. Subsequently, an incision along the midline of the knee was opened through the skin, subcutaneous tissue, and articular capsule of each mouse. The articular capsule was then opened to expose the right knee joint. Next, a 1-mm section of articular cartilage was removed from the distal femur using an electric drill. A sterile prosthesis was then inserted into the intramedullary cavity of the femur using a homemade pointed cone. The incision was then cleaned and sutured. The mice were given intramuscular injection of gentamycin (200 U) continuously for 3 days. After 2 weeks of normal feeding, I/R was performed.Before I/R, the mice in the Sevo group received 3 doses of 2.0% Sevo  at 15 min intervals from a Sevo vaporizer .Mice in the I/R and Sevo groups were then fixed on the operating table in a supine position, anesthetized by intraperitoneal injection of pentobarbital (30 mg/kg) and disinfected. Their femoral arteries were fixed and separated. An atraumatic artery clip was used to block the artery in each mouse for 5 h, and blood flow was restored 12 h later.Two weeks later, the mice were anesthetized by intraperitoneal injection of 2% pentobarbital sodium. Subsequently, the knee joint cartilage was removed from each mouse and washed in ice-cold saline to remove the blood. After shearing and grinding, the knee joint cartilage was centrifuged at 3000 r/min for 15 min to prepare a tissue homogenate. Next, the supernatant was collected and stored in a 1.5-mL Eppendorf tube at −20 °C. At the same time, a section of the limb skeletal muscle was collected and stored in liquid nitrogen, while the remaining limb skeletal muscle was fixed in 10% formaldehyde, dehydrated in gradient alcohol for 24 h, and embedded in paraffin.Skeletal muscle tissues collected from four mice in each group were sliced into 5 μm serial sections, followed by extension at 45 °C and 1 h drying at 60 °C. After being dewaxed in xylene and rehydrated, the sections were stained with hematoxylin  for 2 min, washed in tap water for 10 s, incubated for 10 s in alcohol containing 1% hydrochloric acid, and washed for 1 min in distilled water. Subsequently, the sections were counterstained with eosin and washed for 10 s in distilled water, followed by dehydration in gradient alcohol. Finally, the sections were cleared in xylene and mounted in neutral gum. Histological changes in skeletal muscle tissues were observed under an optical microscope .The concentrations of lactic dehydrogenase (LDH) and inflammatory factors, such as tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), in plasma were determined using corresponding ELISA kits . ELISA assays were carried out by strictly following the procedures provided by the manufacturer .2O2 for 10 min and washed with distilled water, followed by antigen repair at high pressure for 90 s. After being cooled to room temperature, the sections were washed with phosphate buffer saline (PBS), blocked with 5% bovine serum albumin (BSA) for 30 min at 37 °C, and then incubated overnight at 4 °C with rabbit anti-mouse KCNMA1 primary antibodies . After PBS washing, the sections were then incubated with biotinylated secondary antibodies (goat anti-rabbit immunoglobulin G (IgG); 1:100, SF8-0.3, Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) at 37 °C for 30 min. Subsequently, after being fully washed, the sections were incubated with horseradish peroxidase (HRP)-conjugated working fluid, stained with 3,3-diaminobenzidine , and counterstained for 5 min with hematoxylin. During this experiment, PBS was used as the negative control. After staining, cells showing a brownish yellow membrane or cytoplasm were determined to be KCNMA1 positive. Five fields of vision were chosen randomly for each section under a high-power lens to detect the localization of positive expression in skeletal muscle cells. The proportion of positive cells was determined to be the positive expression rate. The experiment was conducted 3 times.Sections were dewaxed twice in xylene (10 min each time) and dehydrated in graded ethanol . Subsequently, the sections were placed on a shaker and washed twice in distilled water (5 min each time). Next, the sections were incubated in 3% H® reagent  and grinded in an ice bath to extract total RNA according to the manufacturer’s protocol. The purity and concentration of extracted RNA were measured by ultraviolet (UV) spectrophotometry . RNA with A260/A280 values ranging from 1.8 to 2.0 was adjusted to a concentration of 50 ng/μL and used as a template for the RT reaction performed with a PrimeScriptTM RT Reagent Kit . The RT reactions underwent 15 min reverse transcription at 37 °C and 5 s inactivation of reverse transcriptase at 85 °C. The total volume of the RT reaction system was 10 μL. The primers  using a two-step method. The RT-qPCR reaction conditions included pre-denaturation at 95 °C for 30 s, and 40 cycles of denaturation at 95 °C for 5 s, annealing at 58 °C for 30 s, and extension at 72 °C for 15 s.  Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as internal references for RT-qPCR. The relative mRNA expression of target genes was calculated using the 2−△△Ct method. Each target gene was tested in triplicate wells on the RT-qPCR plates for each sample.Skeletal muscle tissues were treated with 1 mL TRIzolFor RT-qPCR, each experiment consisted of pre-denaturation at 95 °C for 30 s, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s. GAPDH was used as the internal reference for RT-PCR. Subsequently, electrophoresis was conducted, and each experiment was carried out 3 times.Skeletal muscle tissues (30 mg in each group) were ground to a uniform powder at low temperature, washed 3 times with PBS, lysed with protein lysis buffer, incubated on ice for 20 min, and subsequently centrifuged at 12,000 r/min for 20 min. The supernatant was then collected, and protein concentration in the supernatant was determined using a bicinchoninic acid (BCA) protein assay kit . Cells in the logarithmic growth phase were centrifuged at 3000 r/min and 4 °C for 20 min to estimate the packed cell volume (PCV). Subsequently, 20 μL of PCV cells were lysed for 30 min with 100 μL of lysis buffer  and centrifuged at 12,000 r/min and 4 °C for 10 min to collect the supernatant protein for Western blot analysis. The protein samples (50 µg in each group) were dissolved in 2× sodium dodecyl sulfate (SDS) sample buffer, boiled for 10 min, separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was then blocked in 5% skimmed milk at room temperature for 1 h, washed twice with PBS, and then incubated overnight at 4 °C with diluted rabbit anti-mouse primary antibodies against KCNMA1 , PKGII , VASP , VEGF , Bax , Bcl-2 , Cyclin D1 , Cyclin D3 , Cdc 42 , caspase-3 , and PARP . After being washed 3 times in Tris-buffered saline tween-20 (TBST), the membrane was incubated with HRP-labeled goat anti-rabbit IgG secondary antibodies (1:5000) for 1 h and washed 3 times with TBST (5 min each time). An enhanced chemiluminescence (ECL) kit was then used for coloration. After liquid removal, the membrane was exposed to X-ray, and the absorbance of protein bands was analyzed using a gel imaging analysis system. In the analysis, GAPDH was used as the internal reference. Relative protein expression was calculated based on the ratio of the average absorbance of the target protein band to that of the internal reference band. Each experiment was conducted 3 times.7 cells/mL in RPMI 1640  medium containing 15% fetal bovine serum (FBS), seeded in a 6-well plate and cultured at 37 °C and 5% CO2 with saturate humidity. The culture medium was replaced every 2–3 days. After the single-layer cells reached approximately 80–90% confluence, they were passaged.Skeletal muscle tissues were randomly chosen from the normal and Sevo groups, rinsed and placed in clean and dry culture dishes. After being cut into small sections, the samples were sterilized with 0.01 M PBS (pH 7.6), centrifuged at 800 r/min for 5 min at room temperature, and digested with 0.5 mg/mL Type IV collagenase  at 37 °C for 15 min to collect the supernatant. The supernatant was then filtered with 40-μm cell mesh and centrifuged at 800 r/min for 5 min to collect the cells into a pellet. Subsequently, the cells were resuspended to a density of 2.5 × 10Cells in the logarithmic growth phase were divided into a control , a blank group , a negative control group , a MBNL1-AS1 vector group , a si-MBNL1-AS1#1 group , a si-MBNL1-AS1#2 group , a si-KCNMA1#1 group , a si-KCNMA1#2 group , and a si-MBNL1-AS1 + si-KCNMA1 group  group. Lipofectamine (lipo) 2000  was used for transfection per the manufacturer’s instructions. At 24–48 h after transfection, the cells were harvested.5/mL and fixed in 4% polyoxymethylene for 15 min. After being washed thrice with PBS, the cells were treated with 0.5% TritonX 100 for 10 min and blocked with 5% goat serum at room temperature for 1 h. Subsequently, the cells were incubated with primary antibodies  at 4 °C overnight and then washed with PBS. Next, the cells were incubated with secondary antibodies at room temperature in the dark for 1 h. Finally, the cell nuclei were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) for 5 min, and the fluorescence signal in each sample was observed.Cells were seeded onto a 96-well plate at a density of 1 × 10http://www.herbbol.org:8001/lrt/). Human embryonic kidney 293T (HEK-293T) cells were inoculated in a 24-well plate for 24 h and then co-transfected with luciferase reporter gene vectors KCNMA1 (pmiRRB- KCNMA1-3′UTR) + MBNL1-AS1 or siRNA-MBNL1-AS1, respectively. At 48 h after transfection, HEK-293FT cells were harvested, washed 2 times with PBS, and lysed. The luciferase reporter activity was detected using a Dual-Luciferase® Reporter Assay System . The relative luciferase reporter activity was calculated as the ratio of firefly luciferase activity to the luciferase activity of the Renilla internal control. The experiment was repeated 3 times.Putative target genes of lncRNA MBNL1-AS1 were determined using the program lncRNATargets . Subsequently, the products were extracted using a QIAquick Gel Extraction Kit . Purified biotin-labeled MBNL1-AS1 RNA was transcribed in vitro with a MEGAshortscript™ T7 Transcription Kit . Subsequently, we used a Pierce™ Magnetic RNA-Protein Pull-Down Kit  to pull down the proteins that interacted with RNA. Finally, the proteins were detected by Western blot analysis.5 cells/mL, which was then inoculated in a 96-well plate in 100 μL/well of medium. The cells were then cultured at 37 °C and under 5% CO2. At 24, 48, and 72 h of cell culture, 10 μL MTT  solution was added to the cells, which were further cultured for 4 h before the supernatant was discarded. Subsequently, 150 μL dimethyl sulphoxide (DMSO) was added to each well, and the optical density (OD) value of each well was measured by an automatic plate reader  at a wavelength of 490 nm. The experiment was repeated 3 times.At 48 h after transfection, cells from each group were harvested and resuspended in Dulbecco’s minimum essential medium (DMEM) containing 10% FBS to a density of 1 × 102 and 37 °C for 2 h. After being washed thrice with PBS, the cells were fixed in 4% polyoxymethylene and then treated with 0.5% TritonX 100 for 5 min and incubated with an Apollo dye for 30 min. Subsequently, the cells were rinsed 3 times with 0.5% TritonX 100 and methanol and stained with DAPI for visualization and analysis. The experiment was repeated 3 times.Cells were seeded in triplicate onto a 96-well plate. On the following day, complete medium containing 50 µM EdU was added to the cells, which were further incubated at 5% CO6 cells/mL. Subsequently, 1 mL of cells was centrifuged at 1500 r/min for 10 min to collect the pellet, which was then fixed with 70% alcohol overnight at 4 °C. The next day, the cells were washed twice with PBS, and 100 μL of cell suspension was incubated in the dark for 30 min with 50 μg of RNAase-containing propidium iodide . Finally, after filtering the cells with a 100-mesh nylon net, the cell cycle in each sample was analyzed at a wavelength of 488 nm on a flow cytometer .At 48 h after transfection, the cells were treated with 0.25% trypsin, and the cell density was adjusted to 1 × 102 incubator at 37 °C for 48 h, the cells were washed with PBS, centrifuged, and resuspended in 200 μL binding buffer. After adding 10 μL of Annexin V-FITC  and 5 μL of PI, the cells were incubated for 15 min at room temperature in the dark before 300 μL of binding buffer was added to each sample. Cell apoptosis was then analyzed at a wavelength of 488 nm on a flow cytometer .Fluorescein isothiocyanate-labeled Annexin V/PI (Annexin V-FITC/PI) double-staining was used for the detection of cell apoptosis. In brief, after culturing the cells in a 5% COt-test, while comparisons among multiple groups were performed by one-way analysis of variance (ANOVA). A p value of <0.05 was considered statistically significant.Data were analyzed using SPSS 21.0 software . Measurement data were expressed as the mean ± standard deviation. Comparisons between two groups were performed by Initially, we used the GSE21164 microarray dataset from the GEO database to identify a differentially expressed lncRNA, MBNL1-AS1, which was overexpressed in I/R mice after TKA . In comparison with the I/R group, the Sevo group showed decreased concentrations of LDH, TNF-α, and IL-1β (p < 0.05), suggesting that the concentrations of LDH, TNF-α, and IL-1β might be higher in mice with I/R injury after TKA.We also used ELISA to measure the concentrations of LDH, TNF-α, and IL-1β in the plasma of the normal, I/R and Sevo groups. As shown in Table p < 0.05). In comparison with the I/R group, the Sevo group showed elevated KCNMA1 expression (p < 0.05), suggesting that KCNMA1 might be poorly expressed in I/R injury after TKA.To clarify how KCNMA1 was expressed in mice with I/R injury after TKA, we performed immunohistochemistry assays. Results indicating positive expression of KCNMA1 in skeletal muscle tissues are shown in Fig. p < 0.05). Compared with the I/R group, KCNMA1, PKGII, VASP, VEGF, Bcl-2, Cyclin D1, Cyclin D3, and Cdc 42 mRNA expression was increased in the Sevo group, while MBNL1-AS1 and Bax mRNA expression was decreased (p < 0.05).To measure MBNL1-AS1, KCNMA1, and cGMP-PKG mRNA expression in mice with I/R injury after TKA, we performed RT-qPCR. As presented in Fig. p < 0.05). The protein levels of KCNMA1, PKGII, VASP, p-PKGII, p-VASP, VEGF, Bcl-2, Cyclin D1, Cyclin D3 and Cdc 42 were increased in the Sevo group compared with those in the I/R group, but the protein levels of Bax, cleaved caspase-3, and cleaved PARP were reduced (p < 0.05). These results suggested that in the normal group, MBNL1-AS1 was poorly expressed but KCNMA1 was over-expressed and that the cGMP-PKG pathway was activated.To measure the protein levels of MBNL1-AS1, KCNMA1 and cGMP-PKG in mice with I/R injury after TKA, we performed Western blot analyses. As shown in Fig. p < 0.05)  Fig. . Compare05) Fig. . The res05) Fig. . These rTo confirm that the cells used in this experiment were skeletal muscle cells, we used RT-PCR and immunofluorescence tests. Figure p < 0.05). In comparison with the blank and NC groups, MBNL1-AS1 mRNA expression was increased in the MBNL1-AS1 vector group, but Bax mRNA expression was elevated in the MBNL1-AS1 vector, si-KCNMA1#1 and si-KCNMA1#2 groups. In addition, KCNMA1, PKGII, VASP, VEGF, Bcl-2, Cyclin D1, Cyclin D3, and Cdc 42 mRNA expression was also reduced in the MBNL1-AS1 vector, si-KCNMA1#1 and si-KCNMA1#2 groups. Furthermore, KCNMA1, PKGII, VASP, VEGF, Bcl-2, Cyclin D1, Cyclin D3, and Cdc 42 mRNA expression was increased in the si-MBNL1-AS1#1 and si-MBNL1-AS1#2 groups, but the expression of MBNL1-AS1 and Bax was decreased (p < 0.05). In the si-MBNL1-AS1 + si-KCNMA1 group, only MBNL1-AS1 mRNA expression was decreased (p > 0.05). In the subsequent experiment, we used RT-qPCR to assess the effects of MBNL1-AS1 on the mRNA expression of KCNMA1 and cGMP-PKG-related genes . Compared with those in the blank and NC groups, the protein levels of KCNMA1, PKGII, VASP, p-PKGII, p-VASP, VEGF, Bcl-2, Cyclin D1, Cyclin D3, and Cdc 42 were decreased in the MBNL1-AS1 vector, si-KCNMA1#1, and si-KCNMA1#2 groups, but the protein levels of Bax, cleaved caspase-3, and cleaved PARP were increased in these groups. While the protein levels of KCNMA1, PKGII, VASP, p-PKGII, p-VASP, VEGF, Bcl-2, Cyclin D1, Cyclin D3, Cdc 42, cleaved caspase-3, and cleaved PARP were all elevated in the si-MBNL1-AS1#1 and si-MBNL1-AS1#2 groups, the protein levels of Bax, cleaved caspase-3, and cleaved PARP (p < 0.05) were decreased in these groups. These results indicated that the KCNMA1 and cGMP-PKG pathways might be negatively regulated by MBNL1-AS1, while the overexpression of KCNMA1 could increase the expression of cGMP-PKG pathway-related proteins suppressed by the overexpression of MBNL1-AS1.Using Western blot analysis, we also assessed the effects of MBNL1-AS1 on the mRNA expression of KCNMA1 and cGMP-PKG-related genes , cells in the blank, NC, MBNL1-AS1 vector, si-MBNL1-AS1#1, si-MBNL1-AS1#2, si-KCNMA1#1, si-KCNMA1#2, and si-MBNL1-AS1 + si-KCNMA1 groups began to grow faster after 48 h. In comparison with cells in the blank and NC groups, cells in the MBNL1-AS1 vector and si-KCNMA1 groups grew slower but grew faster in the si-MBNL1-AS1#1 and si-MBNL1-AS1#2 groups (p < 0.05), while no significant differences were shown in the si-MBNL1-AS1 + si-KCNMA1 group (p > 0.05). The results of the EdU assay were consistent with those of the MTT assay. In comparison with the blank and NC groups, the cells in the MBNL1-AS1 vector, si-KCNMA1#1, and si-KCNMA1#2 groups grew slower, but the cells in the si-MBNL1-AS1#1 and si-MBNL1-AS1#2 groups (p < 0.05) grew faster, while no obvious differences were found in the si-MBNL1-AS1 + si-KCNMA1 group (p > 0.05).  These results showed that MBNL1-AS1 could inhibit the proliferation of skeletal muscle cells, while KCNMA1 could promote cell growth. In addition, the overexpression of KCNMA1 alleviated the inhibitory effect of MBNL1-AS1 on the proliferation of skeletal muscle cells.Using MTT and EdU assays, we investigated the effects of MBNL1-AS1 on skeletal muscle cell proliferation. As shown in Fig. p < 0.05). In comparison with the blank and NC groups, the number of cells in G0/G1 phase was increased in the MBNL1-AS1 vector, si-KCNMA1#1, and si-KCNMA1#2 groups, but the number of cells in S phase was decreased in these groups. In addition, the number of cells in G0/G1 phase was decreased in the si-MBNL1-AS1#1 and si-MBNL1-AS1#2 groups, while the number of cells in S phase was increased in these groups (p < 0.05). Furthermore, no significant differences were shown in the si-MBNL1-AS1 + si-KCNMA1 group (p > 0.05). Annexin V-FITC/PI staining was conducted to evaluate the effects of MBNL1-AS1 on cell apoptosis , while the apoptosis rate was decreased in the si-MBNL1-AS1#1 and si-MBNL1-AS1#2 groups (p < 0.05). In addition, no significant differences were observed in the si-MBNL1-AS1 + si-KCNMA1 group. These results suggested that MBNL1-AS1 could promote the apoptosis of skeletal muscle cells, while KCNMA1 could inhibit the apoptosis of these cells. In addition, the overexpression of KCNMA1 alleviated the effect caused by MBNL1-AS1 overexpression  channel proteins in the cell membrane, KCNMA1 mainly acts to maintain the intracellular and extracellular balance of K+ and Ca2+, as well as to stabilize membrane potential and regulate the contraction of vascular smooth muscle cells28. Moreover, a previous study demonstrated that KCNMA1 showed a positive effect on the survival of cardiomyocytes after I/R injury, while cardiac BK channels encoded by KCNMA1 could protect against I/R injury through the participation of reactive oxygen species (ROS)29. In addition, the involvement of KCNMA1 in the modulation of excitability in skeletal muscles might explain the oxidative muscle injury and muscle atrophy caused by I/R injury after TKA30. In addition, KEGG enrichment analyses indicated that KCNMA1 was enriched in the cGMP-PKG signaling pathway, which could regulate diverse cellular functions and gene expression patterns34. Such results were in line with a previous discovery showing that testosterone-induced relaxation of porcine coronary arteries relied on the activation of BK channels via the cGMP-PKG signaling pathway35. In summary, we speculated that the expression of KCNMA1 increased by the down-regulation of MBNL1-AS1 opened the BK channels via the cGMP-PKG signaling pathway to protect skeletal muscle cells against I/R injury after TKA.Represented by immune and inflammatory responses, the patterns of lncRNA–mRNA co-expression have also been shown in muscle contraction36. Further studies also demonstrated that extra-matrix LDH is strategically positioned within skeletal muscle fibers to functionally interact with mitochondria and that the suppressed mitochondrial pyruvate supply can inhibit mitochondrial respiration37. A previous study also found that LDH activity is elevated in the plasma during I/R injury, consistent with our results38. In the I/R group, skeletal muscle tissues showed necrotic muscle fibers and inflammatory cell infiltration, signs indicative of serious nuclear deformation and fibrinolysis. Moreover, it is well known that myocytes preconditioned by exposure to a PKG activator (Sp-8-Br-PET-cGMPS) show a marked decrease in DNA and LDH damage, either due to the direct activation of ATP-sensitive K+ channels or via the PKG signaling pathway, thus leading to cardioprotection39. The increased levels of TNF-α and IL-1β, two circulatory markers of inflammation, in the serum at the early phase of reperfusion can further lead to more severe I/R injury through inflammatory response and oxidative stress41.LDH not only promotes the interconversion of pyruvate and lactate but also serves as a key metabolite of skeletal muscle and promotes muscle mitochondrial functionsIn conclusion, our data may explain the mechanism underlying the effect of the down-regulation of MBNL1-AS1 and the subsequent promotion of the proliferation and inhibition of skeletal muscle cell apoptosis, as we found that the down-regulation of MBNL1-AS1 exerted its effect by up-regulating the expression of KCNMA1 and by activating the cGMP-PKG signaling pathway. As a result, the down-regulation of MBNL1-AS1 protected Sevo-pretreated mice against I/R injury after TKA. Although the factors and signaling pathways that regulate the metabolism of skeletal muscle cells are complicated and still poorly understood, we still believe our findings may provide a rational basis for the clinical application of MBNL1-AS1 as a target in the treatment of I/R injury after TKA.Supplementary Table


Two types of intercellular Ca2+ waves that differ in propagation distance and speed were observed. One type was observed to travel up to 100 μm with an average velocity of 7 μm/s. Such waves were initiated by local tissue damage in the outer hair cell region. The propagation distance was decreased when the purinergic receptor antagonists pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid  or suramin (150 μm) were added to the extracellular buffer. Immunocytochemical analysis and experiments with calcium indicator dyes showed that both P2X and P2Y receptors were present in supporting cells. A second class of waves identified to travel longitudinally along the organ of Corti propagated at a lower velocity of 1–3 μm/s. These ‘slow’ Ca2+ waves were particularly evident in the inner sulcus and Deiters’ cells. They travelled for distances of up to 500 μm. The slow Ca2+ signalling varied periodically (approximately one wave every 10 min) and was maintained for more than 3 h. The slow waves were not affected by apyrase, or by the P2 receptor agonists suramin (150 μm) or PPADS (50 μm) but were blocked by the connexin channel blockers octanol (1 mm) and carbenoxolone (100 μm). It is proposed that the observed Ca2+ waves might be a physiological response to a change in extracellular environment and may be involved in critical gene regulation activities in the supporting cells of the cochlea.We have investigated wave‐like cytoplasmic calcium (Ca 2+ waves are increases in cytoplasmic Ca2+ levels that propagate between cells.Intercellular Ca2+ waves have been linked to gene regulation and are thought to play a crucial role in the development of our hearing epithelium, the organ of Corti and the acquisition of hearing.Periodic Ca2+ waves in supporting cells of an ex vivo preparation of the adult mouse organ of Corti, and these waves were found to propagate independently of extracellular ATP and were inhibited by the gap junction blockers 1‐octanol and carbenoxolone.We observed regular periodic intercellular Ca2+ waves in the organ of Corti is not restricted to the prehearing period.Our results establish that the existence of periodic Ca They have been shown to be present in a variety of cell types including glia, vascular endothelial and smooth muscle cells, hepatocytes in vivo, pigment and Müller cells of the retina as well as in immature cochlear supporting cells . The increased [Ca2+]i in turn triggers a release of the intracellular messengers inositol tris‐phosphate (IP3) into the cytoplasm and a release of ATP into the extracellular environment. [Ca2+]i levels are then increased in neighbouring cells via at least two distinct mechanisms: (1) IP3 diffusion into neighbouring cells via gap junctions triggering an increase in [Ca2+]i levels; and (2) ATP release into the extracellular environment activating P2 receptors on neighbouring cells and leading to a repeat of the cycle. The kinetics and propagation of the wave thus depend on multiple steps.One of the mechanisms of such wave propagation involves purinergic P2 receptors , Ca2+ waves have been observed in the mature organ of Corti, although cochlear supporting cells are extensively coupled via gap junctions and express purinergic P2Y receptors involved in Ca2+ wave propagation in immature tissue  Act 1986 and approved by the UCL Biological Services Animal Ethics Committee.m): 140 NaCl, 1.3 CaCl2, 1 mgCl2, 5.4 KCl, Hepes 10. The osmolarity of the extracellular solution was adjusted to 320 mosmol/l with glucose and the pH was adjusted to 7.4 with NaOH. The bone covering the apical turn of the organ of Corti and Reissner's membrane was removed using fine forceps to gain access to the scala media, exposing a small opening to observe the structures below between the 10% and 30% positions along the partition from the apex. In some experiments the tectorial membrane (TM) covering the organ of Corti was gently removed using a glass micropipette while observing the organ under a microscope. The total procedure took no longer than 15 min. This preparation will be termed an ‘in situ’ cochlea. All reagents were obtained from Sigma‐Aldrich  unless stated otherwise.Wildtype [postnatal day (P)15–90] c57BL/6NCrL mice of either sex with normal hearing were killed by cervical dislocation in accordance with UK Home Office regulations. The hearing function of the c57BL/6NCrL mice was not tested systematically, but we note that the wild‐type mice used all responded to a simple acoustic startle sound. Most animals were in the age range P25–P35. The temporal bone containing the semicircular canals and the cochlea was removed and placed in extracellular solution containing (m3), before staining. Primary antibodies against P2X receptors and myosin VIIA were used. These were a P2X2 rabbit polyclonal antibody  and a MYO7A monoclonal antibody, . Secondary antibodies, Alexa Fluor 488 (A‐11034), Alexa Fluor 568 (A‐11011) and Alexa Fluor 555 (A‐21428), were obtained from ThermoFisher, UK. Primary antibodies were used at 1:500 dilution or 1:250 dilution in the case of MYO7A.Cochleas were fixed immediately after dissection in 4% paraformaldehyde for 30 min and stored in PBS containing 0.05% sodium azide  at a concentration of 20 μm for 45 min at 37°C. Fluo4‐AM was used in all experiments apart from those in which external ATP P2 receptor agonists were applied, in which case cells were loaded with OGB1‐AM with the same protocol. Pluronic acid was present at a concentration of 0.04% (v/v). In preliminary experiments we found that loading into supporting cells with Fluo4‐AM occurred more efficiently than with OGB1‐AM. However, both calcium indicator dyes were used interchangeably in subsequent experiments. In some cases, a ‘nominally zero’ Ca2+ (0 Ca2+) solution was used in further steps after incubation, obtained by omitting Ca2+ from the extracellular solution but compensating for the reduced osmolarity. ‘Nominally zero’ 0 Ca2+ was measured to be 60 μm as well as estimated from the specified content of the reagents. In some experiments  using a 100 μs pixel dwell time. This method of permeabilizing cell membranes mimics the approach used previously in the immature organ of Corti : 130 KCl, 0.5 EGTA, 2 mgCl2, 10 Hepes, pH 7.3. Glucose was used to adjust the osmolarity to 320 mosmol/l.Whole cell tight seal recordings were made using an Axopatch 200B amplifier . Data were filtered at 10 kHz and sampled using a 1322A interface (Molecular Devices). Data analysis was performed using pClamp 9.0 software. Recording electrodes were made using 1.2 mm outer diameter thin wall capillaries, pulled so that the resistance in the bath was 3–4 MΩ. The intracellular solution contained  equipped with 10×/0.3w and 60×/0.9w objectives was used for all electrophysiology experiments. Different cells in the organ of Corti could be readily distinguished by their characteristic morphology. The pipettes were guided to identified cells under visual control.A closed loop perfusion system, with a total volume of approximately 500 ml circulated by peristaltic pumps  was set up to provide constant perfusion of samples at a rate of 2.5 ml/min. The extracellular solution in the chamber was kept at a temperature of 30°C, using a custom built temperature controller and heating loop.After obtaining whole cell recording conditions, cells were held at −55 mV. Capacitance, series and membrane resistance were determined from a 20 mV hyperpolarizing step at the start of the experimental runs.m and pressure was applied using a pressure injector  so that the timing could be controlled accurately. In some cases multi‐barrel pipettes were used to avoid unnecessary manipulation and multiple different agonists could be applied to the same section of the organ of Corti. Agonists were applied sequentially allowing at least 300 s between applications to allow the receptors to re‐sensitize.For agonist application, glass pipettes (1.2 mm outer diameter) were pulled to an opening diameter of 1–2 μm. ATP from a concentrated stock was dissolved in extracellular solution to a final concentration of 100 μFluorescence was measured as the average pixel value in 5 × 5 μm regions of interest (ROIs) placed over identified cell types. Data are presented as fractional increases in fluorescence from baseline. ImageJ and Matlab  were used for image processing.x–t plots) were constructed by drawing a curved line along the imaged length of the organ of Corti and measuring the pixel value at every point of this line. Such pixel values were then displayed as ensemble x–t scans. Such kymographic images were used to analyse time‐resolved Ca2+ wave activity along the Deiters’ cell and IS regions. Images were thresholded using the default automatic threshold function in ImageJ, which is the modified IsoData algorithm implemented in ImageJ ver. 1.41. The binary images established the profile of the Ca2+ peaks in the x–t plane. They were used to calculate the Ca2+ wave travel speed (from the slope), the distance travelled (from the uninterrupted length of the trace) and the average interval between Ca2+ waves. To enhance the signal and to show the propagation of the waves, the kymograph series were in some cases constructed from differenced frames separated by six frames (i.e. Δ(n) = image(n) − image(n − 6) for each n), an improvement over differencing adjacent frames.Kymographic images  was assumed when determining the significance of the difference between groups. Kruskal–Wallis ANOVA was used to compare groups when it could not be assumed that the data had a normal distribution.Unless otherwise stated, mean data are shown ± SD. Statistical analysis was carried out such that m ATP in extracellular solution was puff‐applied to adult organs of Corti. [Ca2+]i, measured by OGB1 fluorescence, were found to increase in the IS, outer pillar (PC), Deiters’ (DC) and Hensen's (HC) cells .To determine if purinergic P2 receptors were present in adult mouse cochleas, 100 μ2+ increases were observed in the same population of cells when 100 μm ATP was applied in normal (1.3 mm) and in nominally 0 mm extracellular Ca2+ solution with 2 mm EGTA added . The increase in fluorescence in 0 Ca2+ was significantly lower however. The fractional increase in response to ATP in 0 Ca2+ solution compared to normal Ca2+ was 0.50 ± 0.11 (n = 7 pairs of agonist applications in 3 cochleas) in IS cells, 0.51 ± 0.13 in pillar cells (n = 7), 0.63 ± 0.24 in Deiters’ cells (n = 7) and 0.90 ± 0.37 in Hensen's cells (n = 4) .Cytoplasmic Cam UTP, a P2Y receptor agonist, was puff‐applied in normal extracellular solution. Application of UTP elicited an increase in [Ca2+]i. As a fraction of the signal induced by 100 μm ATP in the same cells, the signal induced by 100 μm UTP was found to be at: 0.67 ± 0.15 in IS cells (n = 13), 0.70 ± 0.10 in outer pillar cells (n = 13), 0.80 ± 0.34 in Deiters’ cells (n = 11) and 0.88 ± 0.23 (n = 10) in Hensen's cells.Similar results were obtained when 100 μRm) between 30 and 660 MΩ  and a zero current potential (V0) between −70 and 9 mV (mean −46.5 ± 19.1 mV). The peak holding current was also found to be variable and at a holding potential of −55 mV lay between 140 and −1300 pA (mean −100 ± 322 pA). The large variability of Rm and holding current of connected cells was probably due to differences in the extent of coupling between patched cells at the start of the recording. It is also reported that changes in turgor pressure exerted through a patch pipette and osmolarity changes can affect cell coupling in the cochlea  activated a peak inward current in IS cells of 556 ± 316 pA . With ATP application, Rm increased but returned towards pre‐ATP exposure levels with time . Thus, 60 s after the start of a prolonged ATP application Rm increased to 1.08 ± 0.92 GΩ . After a further 2–3 min, the subsequent changes in the current‐voltage curve (I–V) showed that cells began to return to a partially coupled state (data not shown). These data indicate that ATP uncouples the gap junctional communication and increases the apparent cell input resistance or equivalently reduces the slope conductance . The large range in Rm values after 60s of ATP application shows that adult IS cells exhibit considerable variability in the susceptibility to ATP‐dependent uncoupling in the cochlea in situ.ATP  although cells still responded to ATP. After uncoupling, the cell input capacitance was in the range 12.7–22 pF, (mean 16.9 ± 3.5 pF) and the zero current potential V0 was between −27 and −14 mV (mean −21.8 ± 6.2 mV).Superfusion of the cochlea with 1 mm ATP evoked a peak inward current of between −150 and −1250 pA  at a holding potential of −55 mV. These evoked currents showed distinct inward rectification .Application of 100 μB). The simplest explanation for the marked resistance change observed in Fig. 2+‐dependent closure of gap junctions. Such observations are consistent with results described in the guinea pig cochlea  was evident in cochlear supporting cells and on the OHC stereocilia . A weak signal was also observed in stereocilia of IHCs . Strong P2X2 receptor staining was observed on the IS cells, border and phalangeal cells, inner and outer pillar cells, Deiters’ cells and Hensen's cells. Between these cells, both types of pillar cells showed the strongest staining at the surface of the epithelium . The results of [Ca2+]i imaging, electrophysiology and immunohistochemistry are thus compatible with the presence of both P2Y and P2X receptors in the adult mouse organ of Corti.P2X2 receptor expression Fig. A,B was 2+ waves’ were observed in confocal time‐lapse recordings from the organ of Corti.Two types of spatially propagating calcium transients or ‘Ca 2+ wave was observed whenever there was a sudden loss of fluorescence in an individual OHC. When this happened, a wave spread out from that point. Individual hair cells acting as the origin could be easily distinguished because, using the calcium dye Fluo4‐AM, the hair cells had a stronger fluorescence signal than the surrounding supporting cells  . Measurements from two cochleas indicate that the average speed of these Ca2+ wavefronts was 6.5 ± 3.4 μm/s (n = 10 waves).The first type of Caet al. 2+ influx. The most economical hypothesis is that such spreading Ca2+ signals were initiated by local release of intracellular ATP as a result of breakdown of the OHC plasma membrane. Addition of the P2 receptor antagonists pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulfonic acid (PPADS) and suramin to the extracellular buffer significantly decreased (P < 0.01) the average propagation distance from 104 ± 41 μm (n = 10 waves) to 52 ± 19 μm (n = 15 waves). There was no significant difference in propagation speed.These waves differ from those observed after direct injury to hair cells in neonatal cochlear explants  from two cochleas.To determine whether such waves could be initiated simply by cell membrane breakdown, rather than mechanical disruption, we used a focused Ti‐sapphire laser pulse to permeabilize cells in the Deiters’ cell region (see Methods). This laser permeabilization method initiated Ca2+ signalling was also observed in these adult cochleas. Slowly propagating increases in intracellular Ca2+ in the organ of Corti  were often found which started spontaneously. As will be seen below these waves propagated without diminution of amplitude and at a lower velocity. The characteristics of slow Ca2+ waves differed significantly from the damage‐induced and spontaneous waves previously described in the immature organ of Corti  repeated periodically.A different type of Carti Fig. A were o2+ waves could be observed separately in the IS region, medial to the IHCs, and in the Deiters’ cell region around the OHCs. Waves terminated after travelling variable distances and the point of termination varied for different waves. The spatiotemporal properties and periodicity of the slow Ca2+ waves were analysed using kymograph displays (see Methods), which were constructed for both the Deiters’ cell and the IS regions . In such a display, the slope of the x–t lines gives the propagated velocity.Waves were observed to initiate at various positions within the organ of Corti and travelled for variable distances usually in excess of 100 μm. Slow Caons Fig. B. In su2+ waves were confined to the Deiters’ cell region. In the absence of the TM but in nominally Ca2+‐free conditions, slow Ca2+ waves propagated in the Deiters’ cell region at a speed of 1.04 ± 0.29 μm/s . This was approximately six times slower than the fast Ca2+ waves described earlier. The mean propagation distance was 148 ± 63 μm  with some waves travelling over 300 μm . When extracellular Ca2+ was increased to 1.3 mm, the slow Ca2+ waves were unaffected: the measured propagation speed was then 1.05 ± 0.56 μm/s and distance 130 ± 46 μm . Thus, external Ca2+ did not affect the propagation properties of these slow waves and in subsequent experiments normal Ca2+ (1.3 mm) was used unless stated otherwise.Depending upon whether the TM, the extracellular matrix overlying the hair cell bundles, was removed or left in place, the pattern of slow wave propagation differed. Removal of the acellular TM had no detectable effect on the quality of fluorescence imaging. When the TM had been previously removed, slow Ca2+ waves were observed not only in the Deiters’ cell region but also in the IS region. In the IS region they travelled at 2.49 ± 0.66 μm/s . This was significantly faster than the wave propagation speed in the Deiters’ cell region under the same conditions . With the TM attached, the waves propagated on average for 136 ± 70 μm in the IS region whereas in the Deiters’ cell region they propagated for 186 ± 127 μm. Thus, both of the these propagation distances were significantly greater than when the TM was removed (P < 0.05).When the TM was left in place and attached, slow CaThe presence of the TM, which rests on the surface of the epithelium, thus affected the speed and wave propagation distance. The most probable explanation is that the TM limits the volume of the extracellular space at the surface of the organ of Corti, the reticular lamina. Thus, diffusion of any signalling molecule away from the apical cell surfaces would be limited. The result suggests that restricted diffusion of a local signal could modulate slow wave propagation.2+ waves, 1‐octanol (1 mm) or carbenoxolone (100 μm) were added to the bath to block junctional communication. In these and subsequent experiments the TM was left in place. Exposure to either of the inhibitors blocked propagation of the slow Ca2+ waves. In the presence of the inhibitors, spontaneous fluctuations in Ca2+ were restricted to individual supporting cells. Without waves there was a somewhat random pattern of Ca2+ fluctuations throughout the tissue .To determine whether gap junctions were involved in propagation of the slow Casue Fig. E.2+ waves depended on ATP receptors, the P2 receptor antagonists PPADS (50 μm) and suramin (150 μm) were added to the bath. With either of these antagonists there was no significant difference in wave travel distance in either the Deiters’ cell or the IS regions .To determine if slow Caons Fig. . In addi2+ waves, we found in a limited number of experiments that slow waves were absent in cochleas from connexin 30 knockout mice, whether the TM was present or not. However, fast Ca2+ waves were observed in in situ cochleas of connexin 30 knockout mice, propagating with the same speed as found in the wild type (data not shown).To further implicate gap junctional communication in the appearance of slow CaB–D shows that at any one place in either the Deiters’ cell row or the IS cell row there was a clear periodicity to the Ca2+ fluctuations, producing a sequence of waves spreading out from the source. To determine the periodicity of the oscillations, the frequency of Ca2+ peaks at a fixed spot on the organ of Corti was measured after the start of the experiment. After the first waves were observed at a particular location, the frequency of slow Ca2+ peaks reached a maximum within approximately 1 h . After onset, the frequency was found to be similar in both the IS and the Deiters’ cell regions reaching 6–7 peaks/h. Thus, for the Deiters’ cell, the mean frequency was 6.3 ± 2.1 waves/h (n = 30 measurements) in normal Ca2+. For the IS cells the mean frequency was 7.3 ± 1.5 waves/h in normal Ca2+ (n = 19 measurements). At any given location, none of the different extracellular solutions used, apart from those containing gap junction inhibitors, significantly affected the frequency of these events.Figure 1 h Fig. A. After2+ waves did not appear immediately after the experiment began, but were obvious within 2–3 h of excision of the temporal bone from the animal. The latency of onset of slow Ca2 waves was also affected by removal of the TM. Thus, without a TM slow Ca2+ waves were found approximately 120 min after the start of the experiment . When the TM remained in place, slow Ca2+ waves appeared later, first being observed approximately after 180 min in both the Deiters’ cell and the IS regions (three cochleas). When the P2 antagonists PPADS (50 μm) and suramin (150 μm) were included in the solutions and the TM was left attached, waves started with a similar delay in both regions.The slow CaB). The decline in OHC survival in the first 1–2 h after the start of observations correlated with the increase in the number of slow Ca2+ waves in those preparations.We quantified the numbers of surviving hair cells in the recordings by using the complete loss of Fluo4 signal as a measure of cell loss. Hair cell numbers began to decline about 2.5 h after death, with only 50% of the hair cells surviving after 5–6 h Fig. B. The d2+ imaging, electrophysiology and immunohistochemistry we have shown that there are propagating Ca2+ waves in the adult mouse cochlea. We found that the purinergic receptors present in the IS of the organ of Corti have properties consistent with ATP‐activated ionotropic P2X receptors. The inward rectification and desensitization of these currents are consistent with those of the mouse P2X2 subtype reported in the mouse organ of Corti  and temperature is known to affect cytoplasmic Ca2+ signalling. For example, in other tissues Ca2+ wave speed has a temperature dependence, Q10, which has been estimated to be between 2 and 3  to P.S. and from the Wellcome Trust to J.F.A (Ref 093084).


The robustness of our methodology lies in the combination of both intensity and polarization measurements of SHG in the overlapping region. This allows the accurate measurement and consequent pixel-by-pixel mapping of the twist angle in this area. For the specific case of 30° twist angle, P-SHG enables imaging of individual layers.Stacked atomically thin transition metal dichalcogenides (TMDs) exhibit fundamentally new physical properties compared to those of the individual layers. The twist angle between the layers plays a crucial role in tuning these properties. Having a tool that provides high-resolution, large area mapping of the twist angle, would be of great importance in the characterization of such 2D structures. Here we use polarization-resolved second harmonic generation (P-SHG) imaging microscopy to rapidly map the twist angle in large areas of overlapping WS In particular, they exhibit unique nonlinear optical properties owing to their reduced dimensionality and lack of centrosymmetry, that give rise to pronounced SHG9. Analysis of the emitted SHG signal provides information on the crystal orientation7 and homogeneity9 as well as the thickness10 and stacking sequence12 of TMD structures. At the same time, there has been an increasing scientific interest in twisted TMD structures that can either occur during chemical vapour deposition (CVD) growth or be prepared artificially. In the latter case, one can tailor the interlayer coupling, that is strongly twist angle–dependent, and thus reveal new physical phenomena16. Therefore, the twist angle can be regarded as a new degree of freedom, enabling tuning of the physical properties of stacked 2D materials. A prominent example was recently reported in a ground-breaking work, showing that bilayer graphene exhibits unconventional superconductivity for a small value of the twist angle between the layers17. It has also been demonstrated that the twist angle allows control of the valley and band alignment of stacked 2D TMDs11 and it results in ultraflat bands and shear solitons in twisted bilayer MoS218, thus enabling ultrafast charge transfer between the 2D layers19. The strain induced SHG20, the twist angle–dependent moiré-templated strain patterning21, the interlayer valley excitons in TMD heterobilayers23, the twist angle-dependent conductivities across MoS2/graphene heterojunctions24, and the moiré excitons in heterobilayers28 are just a few more studies that have also been reported recently. These observations indicate the strong potential to harness and tune the physical properties of layered 2D materials, via the adjustment of the twist angle of the stacked layers29. In this context, the development of an optical technique capable to map the twist angle with high precision over large areas would be an invaluable tool for the construction and characterization of such new materials.The graphene-related atomically thin 2D TMDs show great promise for high-tech optoelectronic applications12. In addition, phase-resolved SHG techniques have also been used for the determination of the relative orientation between monolayers30. In all these cases however, solely variations in SHG intensity were used to identify the armchair angle difference, a criterion that cannot unambiguously exclude other phenomena as a source of these changes, such as structural transformations and inhomogeneities. The novelty of our method lies in the combination of intensity and polarization-resolved SHG measurements in the overlapping region of stacked 2D materials. SHG intensity-only measurements are insufficient for the determination of the twist angle since variations in intensity may also be caused by changes in the stacking sequence of the layers (e.g. from 2 H to 3 R). On the other hand P-SHG modulation may be due to imperfections of the crystal quality that result in local changes of the main crystallographic axis9. Our work aims to resolve this issue by offering, for the first time, a combined SHG intensity and P-SHG study of stacked 2D structures, that allows the pixel-by-pixel mapping of the twist angle.Earlier studies on the SHG interference from artificially stacked TMD bilayers have shown that the differences in SHG intensity can be attributed to differences in the armchair orientation between the two twisted TMD layers2 layers with different armchair orientation, and an artificially dry stacked WS2 twisted bilayer that has been produced with mechanical exfoliation. Interestingly, we show that there is a specific twist angle of 30°, for which the SHG signal originating from the stacked single-armchair layers (SLs) can be selectively switched-on and -off. This enables the effect of optical discrimination of atomically thin layers and therefore provides a form of axial super-resolution SHG imaging of each individual layer of stacked 2D TMDs.As a proof of concept, we demonstrate the advantages of our all optical nonlinear imaging method, by application both to a pair of overlapping CVD-grown WS31. Each one of those SLs is acting as a surface phase array antenna inside the SHG active volume32. In order to examine the effect of the twist angle of the stacked SLs on their combined SHG pattern, we have performed polarization-in, polarization-out SHG measurements on a raster-scanned area of a CVD-grown WS2 sample and an artificially prepared WS2/WS2 bilayer.Within our approach, the SHG emission from a layered TMD is considered as the result of interference between SLs. As a SL is considered a 2D layer of 2 H or 3 R stacking sequenceφ, rotates and that the modulation depends on the armchair orientation. Using this P-SHG modulation one can map with high precision (~0.15°) the armchair angle, θ, at every pixel of the image9. In the case of two SLs that partially overlap, the P-SHG from each individual layer modulates with a phase depending on the armchair orientation of the layer, while in the overlapping area, the P-SHG modulation follows the SHG interference of the two SLs. Based on this effect, it can be shown that upon using appropriate linear polarization for the excitation of layered WS2 with two different armchair angles overlapping in a region, we are able to decompose the interfered SHG signal originating from each individual layer. This is shown in Fig. 2 triangular flake that exhibits a central region of enhanced SHG intensity. In particular, by rotating the linear polarization of the excitation field, incident to the overlapping area of two SLs, we are able to switch–on the SHG signal from one SL, while the SHG signal from the other is switched–off and vice versa. As shown in detail below, such SHG switch–on/off effect occurs when the armchair angle difference, i.e. the twist angle, between the layers equals to 30°.In our recent work on P-SHG of TMDs, we have demonstrated that the SHG signal modulates as the angle of linear polarization of the excitation field, ision ~0.5° the ar2 sample, the crystal could be a mixture of 3 R and 2 H phases33. A direct consequence of such deviation from the ideal stacking sequence is the incomplete constructive or destructive interference of the SHG fields from different layers. Furthermore, the surface dipoles of the SLs are misaligned and the total SHG signal, as well as its polarization, depend on the twist angle. Regardless such crystal structure deviations, here we show that by using P-SHG measurements one can map for each pixel the armchair orientation of each SL constituting the multilayered structure. In order to accomplish that, the measurement utilizes the rotation of the linear excitation polarization with respect to the X-lab axis and a polarization analyzer parallel to X-lab axis, prior to SHG signal detection  is at an angle θ1 with respect to the X-axis, whereas the armchair orientation of the second SL (x2y2z2 coordinate system) is at an angle θ2 with respect to the same axis . This assumption is realistic since layers of the same symmetry and similar composition should possess similar χ(2). The blue polar diagrams shown in Fig. 1 = 1 and θ1 = 0°), whereas the green polar diagrams show the SHG from a second SL , the SHG from SL1 (blue polar) reaches its maximum first, while the SHG signals from both SL2 (green polar) and the overlapping region (red polar) are zero. Subsequently, the SHG from both SL1 and SL2 goes to zero while that from the overlapping region maximizes. Finally, both the SHG from SL1 and the overlapping region go to zero as the SHG from SL2 becomes maximum. This means that in the case of two overlapping layers at Note that for 35).Based on the above analysis, the amplitude of the P-SHG modulation (see Eq. ) originaφ = 64ο shows the SHG signal originating from one SL for the region of interest (ROI) shown in Fig. φ = 17ο presents the SHG from an assumed second SL  within the same layer.Figure (see Eq. ). Finall1 = 39.5°. Using Eq. /2, that θ2 = 2θeff − θ1 = 10.7°, which corresponds to the armchair angle of the second SL that produces the SHG interference (see Video 1 − θeff) = 28.8°.In order to rule out the possibility of a change in the stacking order, intensity-only measurements are not sufficient and one has to perform a P-SHG analysis. Figure sing Eq.  with θ1 eff should be equal to θ1 (3 R stacking occurs in lattices of the same armchair orientation). Indeed, using P-SHG measurements we calculate a θeff that indicates the presence of a second SL with armchair orientation that fits very well with that calculated for the overhanging SL2 region. These results denote a continuous region of the same armchair orientation and thus the presence of a second SL. This region can also be optically isolated in the SHG image of Fig. 29. Therefore, any detected SHG signal that does not comply with this pattern modulation, is excluded during the data fitting.From the above observations we can exclude the possibility of a change in the stacking order within the same SL in the overlapping region, since in that case θδ = 30° required for complete suppression of the SHG signal from one SL. Confirming our theoretical prediction . Using this map one can create the armchair histogram of specific areas, for example the one shown in Fig. ο for the outer region, but only θ ~ 25ο for the central one (yellowish). In addition in Fig. ο (blue). The above results confirm the presence of more than one SL. If we now assume two different SLs, fix the measured θ1 = 39.5° in the monolayer and use the measured θeff in the overlapping region, we obtain the map of the twist angle shown in Fig. δ> = 30.12° and σ = 3.44°.In this context, we can interpret in detail the P-SHG map obtained from the multilayered CVD-grown WS2 homobilayer  = 14.9o, σ = 1.3° /2 which dictates the P-SHG modulation in an overlapping region of two stacked twisted 2D layers. This novel concept of the effective orientation allowed the determination of the armchair orientation of a second layer that contributed to the total SHG signal detected in the overlapping region. Consequently, by determining the crystal orientation of the second layer that resulted to the measured SHG in the overlapping region we were able to calculate the twist angle between the two layers and for the first time create its pixel-by-pixel mapping both in CVD-grown and in artificially stacked WS2 bilayers. Thus, we have demonstrated an all-optical technique that identifies the presence of stacked SLs, calculates and maps their twist angle. In addition, we have shown experimentally and interpreted theoretically, that when the twist angle between the two SLs is 30° one can selectively suppress the SHG from one layer, while at the same time the SHG from the other is switched-on and vice-versa. This SHG “magic” twist angle enables axial super-resolution imaging and consequently provides a quality characterization of the layered 2D-structure. We envisage that our methodology will provide a new and easy characterization tool of twisted 2D crystals towards their numerous optoelectronic applications.In conclusion, we have used P-SHG imaging microscopy to map the twist angle in stacked TMD layers. In particular, the effect of the SHG interference in the overlapping region of two individual WS2/WS2 stacked structure) is performed in the epi-detection using a dichroic mirror , with the forward P-SHG detection module described above, placed in an epi-detection port of the microscope. Coordination of PMT recordings with the galvanometric mirrors for the image formation, as well as the movements of all the motors, is carried out using LabView  software.Our experimental apparatus is based on a diode-pumped Yb:KGW fs oscillator  inserted in a modified Axio Observer Z1  inverted microscope Fig. . The las2 samples were grown by the low-pressure chemical vapor deposition method (LP-CVD) on a c-cut (0001) sapphire substrate (2D Semiconductors). Note that in the case of CVD-grown samples the stacking of layers is not artificial like in12 but occurs naturally during the growth35. Nevertheless, we expect a similar behavior like in35 from CVD-grown layered samples. This effect has been observed previously and is attributed to the nucleation that occurs during the CVD-growth and commences from the center36.The WS2 bulk crystals were exfoliated by micromechanical cleavage on a polydimethylsiloxane (PDMS) stamp, placed on top of a glass slide for optical inspection. The first monolayer was transferred on a Si/SiO2 (285 nm) substrate by an all-dry viscoelastic stamping and then it was mounted on a XYZ micromechanical stage37. The stage was placed under a coaxially illuminated microscope and following the same procedure, a second WS2 monolayer on a different PDMS was carefully aligned and then stamped slowly on top of the first monolayer. The final step included a controlled release of the PDMS stamp for the fabrication of the WS2 bilayers.WSVideoS1VideoS2VideoS3


In this study, we investigated the function of CLIC1 in Ca2+ and ROS signaling in A549 human lung cancer cells. Depletion of CLIC1 via shRNAs in A549 cells increased DNA double-strand breaks both under control conditions and under treatment with the putative anticancer agent chelerythrine, accompanied by a concomitant increase in the p-JNK level. CLIC1 knockdown greatly increased basal ROS levels, an effect prevented by BAPTA-AM, an intracellular calcium chelator. Intracellular Ca2+ measurements clearly showed that CLIC1 knockdown significantly increased chelerythrine-induced Ca2+ signaling as well as the basal Ca2+ level in A549 cells compared to these levels in control cells. Suppression of extracellular Ca2+ restored the basal Ca2+ level in CLIC1-knockdown A549 cells relative to that in control cells, implying that CLIC1 regulates [Ca2+]i through Ca2+ entry across the plasma membrane. Consistent with this finding, the L-type Ca2+ channel (LTCC) blocker nifedipine reduced the basal Ca2+ level in CLIC1 knockdown cells to that in control cells. Taken together, our results demonstrate that CLIC1 knockdown induces an increase in the intracellular Ca2+ level via LTCC, which then triggers excessive ROS production and consequent JNK activation. Thus, CLIC1 is a key regulator of Ca2+ signaling in the control of cancer cell survival.Chloride intracellular channel 1 (CLIC1) is a promising therapeutic target in cancer due to its intrinsic characteristics; it is overexpressed in specific tumor types and its localization changes from cytosolic to surface membrane depending on activities and cell cycle progression. Ca A chloride channel, a membrane protein involved in chloride transport, indirectly regulates calcium signaling to protect cancer cells from free radicals and DNA damage. Hana Cho from Sungkyunkwan University School of Medicine in Suwon, South Korea, and colleagues inhibited the CLIC1 chloride channel in human lung cancer cells and observed increased activity of a calcium channel. This led to elevated levels of calcium ions within the cells, which in turn triggered the production of chemically reactive molecules that both damaged DNA and activated a signaling cascade that promotes cell death. Many different types of cancer express CLIC1 at high levels. The findings show how the chloride channel helps protect tumor cells from surges in calcium and free radicals, and highlight the therapeutic potential of targeting CLIC1 to slow tumor growth. A high rate of proliferation, active migration, and invasiveness into nonneoplastic tissues are specific properties of neoplastic transformation. All these actions require partial or total involvement of Cl- channel activity6. Thus, this class of membrane proteins could represent valuable therapeutic targets for the treatment of resistant tumors. However, drug design targeting ion channels is difficult because of the vital role of these channels for essential physiological functions in normal cells. Considering this difficulty, a new protein family, the chloride intracellular channels (CLICs)—particularly CLIC1—could be a promising class of therapeutic targets because of their intrinsic properties. First, CLIC1 is overexpressed in particular tumor types, such as hepatocellular carcinoma7, gallbladder carcinoma8, gastric carcinoma9, and colorectal cancer11. Second, CLIC1s change their localization from cytosolic to transmembrane as active ionic channels or signal transducers during cell cycle progression in certain cases13. These changes in intracellular localization and channel function, which are associated with malignant transformation, may offer a distinct target for cancer therapy that can likely spare normal cells. Therefore, understanding the role and underlying molecular mechanism of CLIC1 in cellular transformation is important for designing a therapeutic strategy.Recent studies have revealed the role of ion channels in the development of different cancers. Currently, Cl24. Given that changes in the level of reactive oxygen species (ROS) are fundamental for cell cycle progression26 and cancer cell survival27, it was suggested that CLIC1 could regulate ROS production in cancer cells. In fact, the inhibition of CLIC1 channel activity by the CLIC ion channel blocker IAA94 reduces intracellular ROS production during hypoxia-reoxygenation treatment in LOVO cells, human colon adenocarcinoma cells, suggesting that CLIC1 sustains ROS levels28. However, CLIC1 can also function as a negative regulator of ROS in other tumors, since the depletion of CLIC1 using siRNA in human esophageal squamous cell carcinoma induced apoptosis via the JNK pathway, which is strongly associated with excessive ROS production30. Furthermore, the mechanism by which CLIC1 regulates ROS is currently unclear. In this study, we investigated the role of CLIC1 and its molecular mechanism in A549 human lung cancer cells. We found that depletion of CLIC1 with shRNA in A549 human lung cancer cells increased DNA double-strand breaks both under control conditions and under treatment with the putative anticancer agent chelerythrine, with a concomitant increase in p-JNK levels. Intracellular Ca2+ measurements revealed that CLIC1 knockdown in A549 cells induced an increase in both the basal Ca2+ level and chelerythrine-induced Ca2+ signaling. In addition, CLIC1 knockdown greatly increased basal ROS levels, an effect that was prevented by BAPTA-AM, an intracellular calcium chelator. The LTCC blocker nifedipine, as well as the suppression of extracellular Ca2+, restored the basal Ca2+ level in CLIC1-knockdown A549 cells to that in control cells. Taken together, our results demonstrate that CLIC1 knockdown induces an increase in the intracellular Ca2+ level via LTCC, which then triggers excessive ROS production and consequent JNK activation.Multiple studies have shown that CLIC1 plays crucial roles in controlling the cell cycle, apoptosis, proliferation, invasiveness, and metastasisBglII and XhoI sites.The shRNA sequences CLIC1-knockdown 1 (5′- GATCCCCGGGAGTCACCTTCAATGTTACTTCAAGAGAGTAACATTGAAGGTGACTCCCTTTTTA-3′) and CLIC1-knockdown 2 (5′- GATCCCCGATGAAGGTGTCTCTCAGAGGTTCAAGAGACCTCTGAGAGACACCTTCATCTTTTTA-3′) were cloned into the pSuper.retro vector . CLIC1 cDNA was amplified from MEFs and inserted into pEGFP-C1  at the E. coli and cleaved with thrombin to remove the GST domain and were then used to immunize BALB/c mice. Immunized splenocytes were fused with myeloma cells and selected with HAT medium. Cell culture medium from the cloned hybridomas was analyzed with ELISA to identify specific antibodies against CLIC1. The specificity of the antibodies was tested with other CLICs .GST-CLIC1 proteins were purified from A549 human lung carcinoma cells were maintained in RPMI 1640 medium containing 10% FBS. To establish the CLIC1knockdown cell line, pSuper.retro-scrambled shRNA or the pSuper.retro-CLIC1 KD1 or KD2 shRNA constructs were transfected into A549 cells using Lipofectamine  and selected with 0.3 µg/ml puromycin. Cell clones were screened for CLIC1 knockdown by immunoblot analysis. For assessing the subcellular localization of CLIC1, A549 cells were transfected with pEGFP-C1 or pEGFP-C1-CLIC1 plasmids using Effectene . For transient knockdown of CLIC1, 50 nM noncoding region siRNA  or siRNA against CLIC1  was transfected into A549 cells using Lipofectamine RNAiMAX .A549 cells stably expressing pSuper.retro-scrambled shRNA or pSuper.retro-CLIC1 KD1 or KD2 shRNA were stimulated with 50 µM chelerythrine for 24 h. Cells were fixed with 4% paraformaldehyde and permeablized with 0.5% Triton X-100 in PBS. Samples were blocked with 5% BSA in PBS and stained with anti-pγH2AX (Ser140) (Invitrogen). FITC-conjugated goat anti-mouse IgG  secondary antibodies were used. For nuclear staining, Hoechst 33258 was used, and slides were mounted with ProLong Gold antifade mount (Thermo Scientific). Confocal images were obtained using an LSM 710 .Cells were lysed on ice for 30 min in lysis buffer (50 mM Tris-HCl (pH 8.0) 0.1% Triton X-100, 50 mM sodium fluoride, 5 mM sodium pyrophosphate, 1 mM PMSF, 1 mM sodium orthovanadate, and 2 mM leupeptin). After centrifugation, the protein concentration in the supernatant was determined by a BSA kit . Samples were separated by SDS-PAGE and transferred and were then immunoblotted with the following antibodies: p-p38 MAPK (Thr180/Tyr182), p38 MAPK, p-SAPK/JNK (Thr183/Tyr185), SAPK/JNK, and p-Akt (Ser473) from Cell Signaling Technology  and α-tubulin from Sigma-Aldrich .2 (1.8), MgCl2 (0.5), NaH2PO4 (0.5), glucose (11.1), and HEPES (5) and was adjusted to pH 7.4 with NaOH. To make the Ca2+-free NT solutions, CaCl2 was replaced with equimolar MgCl2. Fura 2-AM was obtained from Thermo Scientific, and chelerythrine chloride and bisindolylmaleimide I were obtained from Tocris Bioscience . All other drugs were purchased from Sigma-Aldrich. Stock solutions of the drugs were made by dissolution in deionized water or DMSO according to the manufacturer’s specifications and were stored at −20 °C. On the day of the experiment, one aliquot was thawed and used. The final concentration of DMSO in the solutions was maintained below 0.1%.The normal Tyrode’s (NT) solution contained (in mM) NaCl (143), KCl (5.4), CaClFor the measurement of intracellular ROS levels, the general ROS marker CM-H2DCFDA (Thermo Scientific) was used. Cells were incubated with 20 µM CM-H2DCFDA for 1 h and were then washed with PBS. CM-H2DCFDA fluorescence was measured using confocal laser scanning microscopy.2+-free NT solution at room temperature. For fluorescence excitation, we used a polychromatic light source , which was coupled to the epi-illumination port of an inverted microscope  via a quartz light guide and a UV condenser. Fluorescence intensity was measured via a 40 × objective (Olympus), a charge-coupled device image intensifier camera  and Metafluor software . Dual excitation at 340/380 was used with a 400-nm dichroic mirror, and emitted light was collected with a 450-nm long-pass filter.Cells were incubated with 3 µM Fura-2 AM  for 45 min in NT solution or Cat-test or one-way ANOVA was used to test for significance; P < 0.05 was considered statistically significant.Data are presented as the means ± standard errors of the mean. Student’s 31. Thus, we examined the effects of CLIC1 knockdown on chelerythrine-induced cellular stress in A549 cells. Consistent with the results of previous studies31, treatment with chelerythrine (50 µM) for 24 h increased the level of pγH2AX in control A549 cells, which was further elevated by CLIC1 knockdown , including c-Jun N-terminal kinase (JNK), p38, and Aktlls Fig. . CLIC1 klls Fig. . Both co31, treatment with 50 μM chelerythrine increased the generation of ROS by 132.09% in control cells. The ROS level in CLIC1knockdown A549 cells was not further increased but rather decreased by 50 μM chelerythrine. However, the ROS level was still higher than that in untreated control A549 cells and was comparable to that in chelerythrine-treated control A549 cells  and 212.6 ± 43.0 nM (n = 17), respectively  had little effect on [Ca2+]i in control A549 cells. However, in CLIC1-knockdown A549 cells, chelerythrine triggered a robust increase in the [Ca2+]i level. Based on the peak chelerythrine-induced [Ca2+]i increase, CLIC1- knockdown A549 cells could be divided into two groups. Even in the mild response group, the peak chelerythrine-induced [Ca2+]i increase was higher than that in control cells  for 10~15 min, and [Ca2+]i was measured. Similar to the data obtained with CLIC1-knockdown cells, CLIC1 inhibition by IAA94 treatment greatly increased the basal Ca2+ level and augmented the chelerythrine-induced Ca2+ increase in A549 cells  for 24 h. As shown in Fig. 2+ level and chelerythrine-induced Ca2+ increase in both control and CLIC1-knockdown A549 cells, suggesting that ROS do not contribute significantly to the increase in the Ca2+ level in CLIC1-knockdown cells.To examine whether ROS play a role in the increase in the basal Ca2+]i in A549 cells. CLIC1 knockdown can regulate basal [Ca2+]i either by activating constitutive Ca2+ entry from the extracellular environment or by promoting Ca2+ release from intracellular stores such as the endoplasmic reticulum. Figure 2+ reduced the basal [Ca2+]i in CLIC1-knockdown A549 cells. This suggests that CLIC1 knockdown regulates the basal [Ca2+]i through Ca2+ entry across the plasma membrane. A recent study showed that the LTCC is influenced by the concentrations of intracellular anions such as chloride36. Thus, we examined the possible involvement of the LTCC in the CLIC1 knockdown-induced increase in [Ca2+]i. Figure 2+ level in CLIC1-knockdown A549 cells from approximately 212.6 nM to 47.8 nM, which was not significantly different from that in control A549 cells . Taken together, these data suggest that CLIC1-knockdown in A549 cells causes the dysregulation of Ca2+ signaling, resulting in excess ROS generation and cellular stress. Based on these data, we propose a working hypothesis that CLIC1 is involved in the regulation of Ca2+ homeostasis through noncanonical LTCC function in A549 cells, thereby preventing excessive intracellular levels of Ca2+ and ROS and controlling cellular stress  the suppression of extracellular Ca2+ attenuated the CLIC1 knockdown-exacerbated Ca2+ response, and (2) nifedipine treatment blocked the increase in the basal Ca2+ level and chelerythrine-induced Ca2+ signal in CLIC1-knockdown cells. Since we detected little LTCC activity in both control and CLIC1- knockdown cells using the patch clamp technique, it is likely that the LTCC regulates the Ca2+ level via a noncanonical mechanism in these cells. Several nonselective cation channels such as TRPC1, TRPC3, TRPC4, and TRPC6 have been found in A549 cells40. Thus, the involvement of nonselective cation channels in CLIC1 knockdown-induced Ca2+ signaling is plausible. However, the pharmacological properties of those TRPCs exhibiting insensitivity to nifedipine41 exclude the possible involvement of TRPCs. Therefore, the LTCC is the most plausible candidate for increased basal Ca2+ levels and chelerythrine-stimulated elevation of intracellular Ca2+ in CLIC1-knockdown A549 cells. However, we cannot completely rule out the possible involvement of other nifedipine-insensitive nonselective cation channels. LTCC proteins are expressed in various cancers42 and have both canonical and noncanonical functions37. It has been demonstrated that LTCC proteins control Ca2+ homeostasis and cell migration in the HCT116 colon cancer cell line by a noncanonical mechanism that involves another channel protein, NCX1/338. In addition, the LTCC can also function as a transcription factor regulating the expression of proteins involved in the regulation of [Ca2+]i and cell migration44. Considering the broad cellular localization of CLIC1 proteins, CLIC1 might also modulate LTCC function as a transcription factor. Further studies are required to elucidate the regulatory mechanism of noncanonical LTCC function in CLIC1-knockdown A549 cells.Our study demonstrates that CLIC1 regulates i) is regulated and maintained by a delicate functional balance between the operations of plasma membrane Cl− channels and those of transporters, as well as those of local impermeant anions47. As intracellular chloride homeostasis is critical for many cell functions, including cell signaling transduction49, intracellular chloride might function as a signaling messenger to regulate LTCC directly or indirectly. In fact, previous studies showed that an increase in chloride levels can increase L-type Ca2+ currents36, possibly via two intracellular regions of the LTCC. It is also reported that the replacement of Cl- with various substituting anions influences many Ca2+-mediated processes, including the contractility of cardiac and skeletal muscle, hormone secretion, and neurotransmitter release via the LTCC54. Thus, it is plausible that CLIC1 knockdown in A549 cells induces an increase in the intracellular level of chloride, which in turn enhances LTCC function. However, the detailed mechanisms by which intracellular chloride regulates the LTCC need to be further investigated.The reduction in the chloride channel activity in cancer cells induces an increase in the intracellular chloride level due to a reduction in chloride efflux2+ signaling can influence the cellular generation of ROS. Interactions between ROS and Ca2+ signaling can be bidirectional, wherein ROS can regulate cellular calcium signaling, while calcium signaling is essential for ROS production55. However, the antioxidant Trolox did not alter the basal Ca2+ level or chelerythrine-induced Ca2+ response in either control or CLIC1-knockdown A549 cells, suggesting that these Ca2+ signals are independent of ROS in A549 cells. These data further support the idea that the mutual interplay and crosstalk between Ca2+ and ROS is highly dependent on the cellular context56. The role of Ca2+ and ROS during the process of apoptosis has been explored in great depth. Ca2+ signals regulate ROS by modulating several ROS generation systems, including NADPH oxidases (Nox), NO synthase (NOS) and mitochondria, and the consequent Ca2+ and ROS surges are required for apoptosis initiation at the mitochondria-endoplasmic reticulum interface57. However, this interplay is altered in cancer cells, enhancing their resistance to apoptosis, but its underlying mechanisms are still unclear57. Taken together with the fact that the activity of JNK and p38 is regulated by intracellular Ca2+ as well as ROS59, our results suggest that the disturbance in intracellular Ca2+ signaling combined with elevated ROS levels might underlie the stronger but more transient activation of MAPK upon chelerythrine treatment in CLIC1 KD1 cells relative to that in control cells and that these surges in Ca2+ and ROS might lead to a cellular stress-induced response and death, implying that CLIC is important for the apoptosis resistance of A549 cells.Our results demonstrated that BAPTA-AM suppressed ROS production in CLIC1-knockdown A549 cells, suggesting that Ca60. However, CLIC1 knockdown in human esophageal squamous cell carcinoma induced apoptosis through the JNK pathway, likely reflecting excessive ROS production61. Similar to its effect in human esophageal squamous cell carcinoma, CLIC1 knockdown in A549 human lung cancer cells upregulated cell death and JNK activation concomitant with the elevated ROS levels. The basis for the different effects of CLIC1 inhibition in distinct cancer cells is currently unclear; however, one of the mechanisms might be the diverse regulatory crosstalk between Ca2+ and ROS56. We demonstrated that CLIC1 inhibition upregulated ROS levels by increasing intracellular Ca2+ through the LTCC in A549 cells. Considering that the functional expression of the Ca2+ signaling machinery, such as the LTCC, varies depending on cell type, downstream signaling effects, such as ROS regulation by CLIC1 inhibition, can vary in different cell types. Further studies are required to clarify these uncertainties. However, our data reveal that CLIC1 might play a critical role in apoptosis resistance, diminishing the large surges in the Ca2+ concentration and ROS levels, and suggest the possibility for targeting CLIC1 to control apoptosis in cancer cells.It appears that the regulation of ROS levels by CLIC1 is tumor cell-type specific. It has been previously shown that the inhibition of CLIC1 by IAA94 significantly suppressed ROS generation in glioblastoma cancer stem cells and LOVO cells, a human colon adenocarcinoma cell lineSupplementary FiguresSupplementary TableSupplementary Figure Legend


The channel is cooperatively affected by vacuolar Ca2+ , suggesting that it may accommodate a Ca2+ receptor that can bind two calcium ions. Alanine scanning of six negatively charged amino acid residues in the transmembrane S5 and S6 linker, facing the vacuolar lumen, revealed that two aspartate residues, 401 and 405, are essential for current inhibition and direct binding of 45Ca2+. Expressed in HEK-293 cells, a significant fraction of TRPY1, present in the plasma membrane, retained its Ca2+ sensitivity. Based on these data and on homology with TRPV channels, we conclude that D401 and D405 are key residues within the vacuolar vestibule of the TRPY1 pore that decrease cation access or permeation after Ca2+ binding.By vacuolar patch-clamp and Ca  •2+The yeast vacuolar TRPY1 channel is inhibited by vacuolar Ca•2+-mediated inhibitionAspartate residues D401A and D405A are essential for Ca•2+ bindingAspartate residues D401 and D405 are essential for direct Ca•2+ binding to D401 and D405 within vacuolar pore vestibule mediates inhibitionCa Structural Biology; Protein Structure Aspects; Biophysics By mediating cation influx, TRP channels shape the membrane potential and increase the cytosolic Ca2+ concentration ([Ca2+]cyt), translating environmental and endogenous stimuli into cellular signals. Most TRP channels fulfill their physiological function in the plasma membrane as cation influx channels, whereas some functional TRP channels are localized in the membrane of cytoplasmic organelles  ion channel family is encoded by more than 100 genes . With thganelles .2+-impermeable TRPM4 and TRPM5 - and also potentiate, activate, or inhibit currents most probably by interfering with TRP channel domains facing the cytosol (YVC1) gene in Saccharomyces cerevisiae and localized in the vacuolar membrane, is activated by Ca2+ interfering with cytosolic channel domains , glutathione, or β-mercaptoethanol  activates additional inward and outward currents (max in Ca), which are absent in vacuoles isolated from YVC1 knockout cells   increased currents in a concentration-dependent manner with apparent EC50 values of 498 μM for the inward current and 391 μM for the outward current  D. In conmplitude E, red IVof 10 μM A and 2B, current B.Figure 2+], we perfused the vacuole with 1 mM Ca2+ by the patch pipette. The currents activated by 1 mM [Ca2+]cyt were significantly reduced  and inhibition (when present at the vacuolar site) (2+ ([Ca2+]vac), cytosolic concentrations of 1 mM divalent cations increased activation in the order Sr2+ (12%) < Mn2+ (15%) < Ba2+ (63%) with 100% current activation at 1 mM [Ca2+]cyt , vacuolar concentrations of 1 mM divalent cations inhibited in the order Mn2+ (33%) < Ba2+ (63%) < Sr2+ (68%) < Ca2+ (96%) (Srar site) . In the Ca2+]cyt A and S2B2+ (96%) C and S2D2+, we expressed the YVC1 cDNA in HEK-293 cells. As shown by western blot the TRPY1 protein is present in transfected cells and a significant fraction is detectable in the plasma membrane by surface biotinylation  or presence of 1 mM vacuolar Ca2+. As shown for vacuoles of wild-type yeast cells  in single-copy pRS316 CEN plasmids without its own promoter. Under these conditions, cytosolic Ca2+-induced vacuolar currents were recorded from YVC1 wild-type and YVC1D401A- and YVC1D405A-transformed YVC1 KO yeast . The unchanged single-channel current amplitude  did not inhibit hyperosmotic shock and cytosolic Ca2+-induced activity of the mutant channels TRPY1D401A and TRPY1D405A  peptides derived from wild-type (amino acid residues F390 to L414) and D401A and D405A mutant S5-S6 linkers of TRPY1 were synthesized and spotted onto hardened cellulose membranes and incubated with 45Ca2+  no detectable 45Ca2+ binding. As control, we spotted a 25-mer peptide representing the first EF hand (EF1) of mammalian calmodulin , additionally estimated by UV absorption at 312 nm , that it requires two aspartate residues (D401 and D405) in the S5-S6 linker facing the vacuolar lumen, and that these aspartate residues are directly involved in Ca2+ binding. The yeast vacuolar TRP cation channel TRPY1 may be assumed as a progenitor of TRP channels present in mammals, flies, and worms and underlies cation movements across the yeast vacuolar membrane. Additional TRP-related proteins in Saccharomyces cerevisiae like FLC2, encoded by the YAL053W gene, seem to be involved in endoplasmic reticulum Ca2+ release  and 391 μM (outward current) with Hill coefficients < 1. Reducing agents such as DTT or β-mercaptoethanol lower the [Ca2+]cyt required for channel activation (2+]cyt of 10 μM of cytosolic Ca2+ (2+]cyt has been estimated to be in the range of 260 nM (2+, which in turn facilitates TRPY1 activity. Palmer et al. (2+ specific, but we also detected activation by the alkaline earths Ba2+ > Sr2+ and by the transition metal ion Mn2+ (We performed whole-vacuole patch-clamp recordings and Cat strain  and HEK- release . TRPY1 i release , reducin release , aromati release , and cytease (2+ . In the tivation , and thetivation A has beetivation . Reducintivation , which mlic Ca2+ , but thef 260 nM . Probablr et al.  suggesteion Mn2+ .2+] within the vacuolar lumen presumably inhibits TRPY1 currents (2+ added to the bath eliminates TRPY1 single-channel currents in inside-out excised vacuolar patch-clamp recordings (2+, as well as Sr2+ and Ba2+, strongly inhibit TRPY1 activity and that the amino acid residues D401 and D405 located in the vacuolar loop between S5 and S6 (2+-dependent inhibition and direct Ca2+ binding. Mutation of the nearby G402 (2+ increased TRPY1 currents in vacuolar excised patches (2+ might be attracted to a single negatively charged residue and thereby prevent efficient coordination of Ca2+. In mouse TRPV3 and TRPV4, corresponding residues D641 and D682, respectively, were shown to be key residues for extracellular Ca2+-mediated channel inhibition (D425 (D401 and TRPY1D405 are located in the vacuolar vestibule of TRPY1's ion-conducting pore. The residues E600, D601, E610, and E648 are involved in pH-dependent modulation of human TRPV1 (D601 aligning to TRPY1D401 (2+-mediated inhibition of TRPV1 and TRPV6, their S5-S6 linkers appear closer related to TRPY1 than TRPV3, TRPV4, TRPV2, and TRPA1 by sequence alignment (D401, are shown to bind and guide Ca2+ into the channel pore of TRPV6 (High , which inhibits TRPY1 (2+-dependent inhibition of TRPML1V432P, and an electronegative luminal pore domain was indicated as a unique hallmark of TRPML1's interaction site for lysosomal Ca2+ and H+ (The physiological pH value in yeast vacuoles is slightly acidic . Vacuolaremained . The mam cytosol , and TRP+ and pH . Like fof 270 μM , which its TRPY1 . Externa+ and H+ .2+ concentration was estimated to be 1.3 mM (2+ store with TRPY1 as Ca2+ release channel (2+, but a considerable amount of Ca2+ is bound by vacuolar polyphosphate (2+] of ∼30 μM (+ and Na+ are the major charge carriers used to record TRPY1 currents in the yeast vacuole and in HEK-293 cells, Ca2+ storage and release may not represent major functions of vacuoles and TRPY1 channels. Instead, upon hypertonic shock, which activates TRPY1, cation efflux from the vacuole might provide osmolytes to the cytosol and thereby counteracts water deprivation. The vacuolar Ca2+-dependent inhibition of TRPY1 might protect the yeast cell from cytosolic Ca2+ overload after hyperosmotic-shock-induced channel activation and increase of the vacuolar [Ca2+] subsequent to loss of vacuolar water.The yeast vacuolar Cae 1.3 mM  and the  channel . Accordihosphate  and therf ∼30 μM  would alf ∼30 μM . Conside2+-dependent modulation of the yeast vacuolar TRP channel and identified two aspartate residues in the outer vestibule as essential for Ca2+ binding and Ca2+-dependent inhibition. The structure of TRPY1 is not yet known, and we aligned the S5, pore linker, and S6 structure of TRPY1 according to the published structure of Xenopus TRPV4. Thus the mechanism we discuss for the Ca2+-dependent inhibition of TRPY1 reflects our experimental data along with a TRPY1 model, obtained due to an alignment to the available structure of another TRP protein. The suggested scenario awaits further support by structural data of TRPY1.We studied the CaAll methods can be found in the accompanying