Idelalisib is approved for the treatment of relapsed chronic lymphocytic leukemia together with Rituximab and for monotherapy of follicular B-cell non-Hodgkin’s lymphoma and small lymphocytic lymphoma. It is a potent and selective phosphatidylinositol 3-kinase-δ (PI3K-δ) inhibitor. PI3K-δ primarily is expressed in B-cells and prevents effectively proliferation in malignant B-cells.We provide a detailed report on treatment history and photo documentation of acute adverse effects of radiation therapy with simultaneous Idelalisib medication in one case of B-CLL. Radiosensitivity tests were performed for the index patient under Idelalisib and after the addition of Idelalisib to healthy individuals’ blood. Radiosensitivity in human lymphocytes was analyzed with a three color in situ hybridization assay. Primary skin fibroblasts were studied after a treatment with Idelalisib for apoptosis, necrosis and cell cycle using flow cytometry. DNA double-strand break repair was analyzed by γH2AX immunostaining.p = 0.05).The index patient presented a strong grade 2 radiodermatitis and grade 3 mucositis after irradiation with 20 Gy and a simultaneous intake of Idelalisib. Irradiations without Idelalisib medication were well tolerated and resulted in not more than grade 1 radiodermatitis. The index patient under Idelalisib had a radiosensitivity of 0.62 B/M which is in the range of clearly radiosensitive patients. A combined treatment of lymphocytes with 2 Gy and 10 nmol/l Idelalisib showed a tendency to an increased radiosensitivity. We found a clear increase of apoptosis as a result of the combined treatment in the Idelalisib dose range of 1 to 100 nmol/l compared to solely irradiated cells or solely Idelalisib treated cells (A combined Idelalisib radiotherapy treatment has an increased risk of side effects. However, combined therapy seems to be feasible when patients are monitored closely. Idelalisib is a potent and selective phosphatidylinositol 3-kinase-δ (PI3K-δ) inhibitor. It is approved for the treatment of relapsed chronic lymphocytic leukemia together with Rituximab and for the monotherapy of follicular B-cell non-Hodgkin’s lymphoma and small lymphocytic lymphoma . PI3K-δ 2 incubator.SBL5 are primary skin fibroblasts derived from a healthy 20-year-old Caucasian male. Fibroblasts were grown in Ham’s F-12Medium  supplemented with 20% fetal bovine serum, 3 mmol/l L-glutamine and 1% penicillin/streptomycin. Lymphocytes were derived ex vivo from a healthy 29-year old Caucasian female and were short time cultured in RPMI supplemented with 10% fetal bovine serum. All cells were cultured at 37 °C in a 5% COCells were irradiated at 120 kV by an X-ray machine  with IR doses of 2 Gy. Unirradiated samples were processed along with irradiated samples.Apoptosis and necrosis were detected by the Annexin-V-APC (Annexin-V-Allophycocyanin) and 7AAD (7-Aminoactinomycin) assay . Briefly, cells were suspended in Ringer solution and stained with Annexin-V-APC (dilution 1:40) and 7AAD (dilution 1:40) for 30 min at 4 °C. Cell cycle analysis was performed by Hoechst 33342 staining. Cells were incubated for at least 1 h in ethanol at 4 °C and staining was performed by Hoechst 33342 (60 μmol/l) in Ringer solution. Flow cytometric measurements were performed with Gallios Cytometer  using 25.000 cells per run. Results were analyzed with Kaluza Flow Cytometry Analysis 1.5a . Each experiment was performed three times with three replicates per run.SBL5 primary fibroblasts were seeded on cover slips in Quadriperm vessels and allowed to adhere for at least 2 days prior to each Idelalisib treatment. Cells were irradiated as described and fixed for 15 min in 4% paraformaldehyde and permeabilized in 0.1% Triton X-100, blocked in 10% fetal calf serum in Tris buffered saline over night at 4 °C. Cells were incubated for 2 h at room temperature with anti-γH2AX  and anti-PML . After washing in TBS, cells were incubated with anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 594 secondary antibodies  for 1 h at room temperature. Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and mounted in Vectashield .Whole blood sample of the index patient was drawn 3 months after the end of the penultimate radiation therapy. One part of the sample was irradiated with a dose of 2 Gy, the other part stayed mock irradiated. The blood was cultivated and lymphocytes stimulated with Phytohemagglutinin. After repair time of 48 h chromosomes were arrested during metaphase by colcemid and prepared on slides. Chromosome 1, 2 and 4 were stained with chromosome specific probes. Aberrations of at least 100 metaphases were analyzed regarding the amount of double strand breaks. The breaks per metaphase (B/M) value of the irradiated sample was then corrected by the background of the mock irradiated attempt (2 Gy corr.). An increased radiosensitivity is assumed with a resulting value of 0.55 B/M or higher –8. For cWhole blood samples were drawn from a healthy 29-year-old Caucasian female. The samples were incubated ex vivo with different concentrations of Idelalisib  for 4 h. One part of each attempt was then irradiated with 2 Gy, whereas the other part stayed mock irradiated. Lymphocytes were then stimulated, chromosomes prepared and stained as described before. The experiment was repeated for at least three times.Mann-Whitney U Test was used for data analysis.The initial diagnosis was set in 2003 with a clinical manifestation of generalized lymphadenopathy. After histological diagnosis a treatment with Chlorambucil and monthly immunoglobulin substitution was started. After a long period of clinical regression, a recurrent disease of the right frontoparietal skin was histologically proven in 2012. 6 cycles of R-Bendamustin-scheme were applied and a radiation therapy to the persisting lesion was performed. 36 Gy in 18 fractions were applied and well tolerated. No significant adverse effect to mucosa and the skin were observed. Complete hematologic remission could be maintained until February 2014, when a local lymphoma manifestation of the larynx with stenosis of hypopharynx was histologically proven. Eight cycles of R-CHOP followed and were well tolerated. In November 2014 a recurrence of the right lower leg and shortly later in January 2015 an extensive lymphoma manifestation including multiple both-sided paranasal sinuses  as well as the ventral base of the skull, orbital, perioral and frontal infiltration were diagnosed.A systemic therapy with 8 courses Rituximab and Idelalisib 150 mg twice daily was started in January 2015 Fig. . In JulyDue to increasing pain in December 2015 radiotherapy with 36 Gy in 18 fractions was applied to the manifestation of the right lower leg. Idelalisib still remained paused during the radiation of the leg. Here a radiodermatitis grade 1 was registered. Idelalisib was re-started in January 2016 with reduced dosing of 100 mg twice daily and was discontinued at the end of March 2016.In a clinical visit end of February 2016 the patient reported well-being, but a weak general condition. The CT scan showed persisting swelling especially of the right sphenoidal and frontal sinus and the left maxillary sinus. A radiological discrimination between persisting tumor and post therapeutic affection was not possible. Histological confirmation was not initiated. Concerning radiation toxicity, there was no more persistence of radiation dermatitis of the facial skin Fig. . The patIn March 2016 radiotherapy was applied to the hypopharynx and retro sternum up to a dose of 27 Gy in 14 fractions. The therapy was interrupted due to a very poor physical condition of the patient; he was transferred to a hospice. There he died in May 2016 as a result of a general progress of the disease.Radiosensitivity in lymphocytes was analyzed with a three-color fluorescence in situ hybridization assay Fig. -c. A comp < 0.05)  Fig. . There i05) Fig. . Likewis05) Fig. -g nor inOne patient suffering from B-CLL was treated with four different radiotherapeutic treatments. He experienced distinct increased side effects of grade 2 dermatitis and grade 3 mucositis after a combined RT-Idelalisib treatment. The dermatitis and mucositis already appeared after a 2 Gy fractionated total dose of 20 Gy. There were no side effects caused by one RT prior to Idelalisib treatment and one RT upon completion of Idelalisib. The radiosensitivity of the index patients using the three-color fluorescence in situ hybridization analyzes showed a clear increase of the radiosensitivity level in index patients under Idelalisib. In primary skin fibroblasts a distinct increase of apoptotic rate was found. Summarizing these data, it is a strong indication that Idelalisib can trigger severe acute side effects by RT .A Limitation of this study could be the fact that the patient suffered from parapsoriasis. However, both RTs prior and post Idelalisib treatment were well tolerated with doses higher than the 20 Gy –radiotherapy which caused the grade 2 dermatitis and grade 3 mucositis. Besides, it must be taken under concideration that we measured an increase of radiosensitivity during Idelalisib treatment but the existing rediosensitivity without Idelalisib is unknown as we could not perform the respective testing. Yet the addition of 10 nmol/l Idelalisib to a healthy individuals blood increased the radiosensitivity.In patients’ blood a concentration of 0.7–10 nmol/l Idelalisib is observed [It is noteworthy that combined treatment of IR and Idelalisib induces a selective induction of apoptosis in fibroblasts. There is only very limited influence on DNA dsb-induction or repair, on cell cycle and on necrosis. This hints to influence of Idelalisib on apoptosis through a specific pathway. Idelalisib inhibits in B-cells the p110δ catalytic subunit of PI3K-δ very specificly . It induIf a combined treatment of Idelalisib and IR is indicated, the high risk of side effects should be considered thoroughly. The main reason for concomitant treatment is the concern for an intensified tumor growth during the time, when the drug is omitted. Yet the acute side effects were manageable. Patients under treatment with Idelalisib should be monitored more particularly to detect and treat toxicities early enough. They should be provided with supportive care similar to patients receiving simultaneous radiotherapy and a BRAF inhibitor or other multikinase inhibitors –16. NeveIdelalisib is radio sensitizing. In combination with radiation it clearly shows a specific induction of apoptosis in cell cultures. A combined Idelalisib radiotherapy treatment has an increased risk of side effects. Facing them, a pause of Idelalisib medication during radiotherapy should be taken into consideration. However, combined therapy seems to be feasible when patients are monitored closely during and after radiotherapy.


Background/Objective: The cardiac Na+/Ca2+ exchanger (NCX) has been identified as a promising target to counter arrhythmia in previous studies investigating the benefit of NCX inhibition. However, the consequences of NCX inhibition have not been investigated in the setting of altered NCX expression and function, which is essential, since major cardiac diseases  exhibit NCX upregulation. Thus, we here investigated the effects of the NCX inhibitor SEA0400 on the Ca2+ transient amplitude and on proarrhythmia in homozygous NCX overexpressor (OE) and heterozygous NCX knockout (hetKO) mice compared to corresponding wild-types (WTOE/WThetKO).Methods/Results: Ca2+ transients of field-stimulated isolated ventricular cardiomyocytes were recorded with fluo-4-AM or indo-1-AM. SEA0400 (1 μM) significantly reduced NCX forward mode function in all mouse lines. SEA0400 (1 μM) significantly increased the amplitude of field-stimulated Ca2+ transients in WTOE, WThetKO, and hetKO, but not in OE . SEA0400 (1 μM) significantly reduced the number of proarrhythmic spontaneous Ca2+ transients (sCR) in OE, but increased it in WTOE, WThetKO and hetKO  and induced Ca2+ overload with subsequent cell death in hetKO.Conclusion: The effects of SEA0400 on Ca2+ transient amplitude and the occurrence of spontaneous Ca2+ transients as a proxy measure for inotropy and cellular proarrhythmia depend on the NCX expression level. The antiarrhythmic effect of SEA0400 in conditions of increased NCX expression promotes the therapeutic concept of NCX inhibition in heart failure/atrial fibrillation. Conversely, in conditions of reduced NCX expression, SEA0400 suppressed the NCX function below a critical level leading to adverse Ca2+ accumulation as reflected by an increase in Ca2+ transient amplitude, proarrhythmia and cell death. Thus, the remaining NCX function under inhibition may be a critical factor determining the inotropic and antiarrhythmic efficacy of SEA0400. Due to these unsatisfying results, much effort has been made to identify alternative targets to prevent from life threatening arrhythmias. In this regard, heart failure—the most prevalent precondition for sudden cardiac death—is accompanied by an upregulation of the cardiac Na+/Ca2+ exchanger (NCX) . Thus, increased NCX function is regarded as a crucial factor for increased triggered activity and whole heart arrhythmia   as previously described with minor alterations  and Protease from streptomyces griseus type XIV (Sigma Aldrich).Ventricular cardiomyocytes were isolated from OE, hetKO and respective wild-type mice  mounted to an inverse microscope . Cardiomyocytes were transferred to a custom-made recording chamber and field-stimulated by platinum wires . Fluorescence signals were recorded by FeliX 1.42 software . The following external solution (Tyrode's solution) has been applied; in mM: NaCl 140, KCl 5.8, KH2PO4 0.5, Na2HPO4 0.4 MgSO4 0.9, HEPES 10, glucose 11.1, CaCl2 2, pH = 7.3. Experiments were performed at room temperature. The NCX function was assessed by the decay of caffeine (10 mM) induced Ca2+ transients . The amplitude of field-stimulated Ca2+ transients served as a proxy measure for inotropy. The most chosen SEA0400 concentration is 1 μM . Numerical results are expressed as mean value ± standard error of the mean (SEM). Number of replicates are indicated as 2+ transient decay indicates a higher NCX function.To test the inhibitory effect of SEA0400 on the NCX forward mode, caffeine application experiments were conducted as reported previously  was significantly prolonged in heterozygous hetKO and shortened in OE as compared to the corresponding WT confirming a reduced/enhanced NCX-mediated Ca2+ removal rate, respectively   . Notably, the mean T50 value of OE in presence of SEA0400 was larger than in WTOE/WThetKO but still smaller than in hetKO under basal conditions indicating a remaining NCX function in OE ranging between the levels of WTOE/WThetKO and hetKO.As demonstrated previously, in absence of SEA0400 , the time to 50% decay of caffeine-induced Ca Figures . When ca Figures . In pres2+ transients, Ca2+ transients were measured in absence and presence of 0.3 and 1 μM SEA0400. Cells were field-stimulated at 1 Hz for 4 min followed by 20 sweeps on 0.125 Hz. This protocol was repeated in presence of 0.3 and 1 μM SEA0400 within the same cell. The amplitude of the Ca2+ transient was quantified at the end of the 1 Hz pacing phase after reaching a steady-state in Ca2+ transient kinetics. The Ca2+ transient amplitude in presence of SEA0400 is given in % of basal conditions.To test the effects of SEA0400 on the steady-state amplitude of field-stimulated Ca2+ transient amplitude in WTOE (n = 27/7), WThetKO (n = 19/4), and hetKO (n = 19/5). However, in OE (n = 27/8) no increase of the Ca2+ transient amplitude was observed under application of SEA0400  , defined as spontaneously occurring Ca2+ transients between the field stimulated Ca2+ transients, during the 0.125 Hz pacing period in the above stated protocol before and after SEA0400 administration. An increase of sCR suggests an increase of proarrhythmia.To investigate the dependence of the antiarrhythmic effects of SEA0400 on NCX expression level, we monitored the occurrence of spontaneous Ca2+ transients was significantly increased in OE compared to WTOE . The average number of sCR per cell .Under basal conditions, the occurrence of sCR Figure  was simiE Figure   as well as mean number of sCR per cell .In hetKO the fraction of cells with sCR Figure   without altering the number of sCR-positive cells . In hetKO application of 1 μM SEA0400 significantly increased both, the occurrence of sCR per cell  and the number of sCR-positive cells . Moreover, SEA0400 drastically impaired Ca2+ homeostasis in hetKO as reflected by an abrupt elevation of the diastolic Ca2+ level, incessant Ca2+ oscillations and subsequent cell death, defined as Ca2+ hypercontraction  but unaltered between OE and WTOE .Application of SEA0400 altered the occurrence of sCR, but the specific effect depended upon the basal NCX expression level. In OE, SEA0400 significantly decreased the mean number of sCR per cell Figure   has been identified as a potential target. Due to its electrogenic mode of action (3 Na+: 1 Ca2+) NCX mediates membrane depolarization during Ca2+ extrusion. Hence, NCX may contribute to the development of EADs and facilitate the translation of spontaneous SR Ca2+ releases into DADs, which both may trigger arrhythmia  and hetKO but interestingly not in OE.SEA0400 reduced the NCX forward mode function to a similar extent in all mouse lines thus confirming inhibitory efficacy. At the same time, SEA0400 mediated a significant increase of the Ca2+ handling and basic electrophysiological properties between the various species. Analogous to cardiac glycosides, NCX inhibition can result in a net gain of intracellular Ca2+, which augments the Ca2+ transient amplitude and consequently inotropy. SEA0400 seems to mediate such a positive inotropic effect in smaller rodents (mouse and rat) but not in higher mammals, with the exception for pigs. In smaller rodents, the intracellular Na+ level is elevated compared to higher mammals which results in a reversal potential for NCX (ENa/Ca) close to the diastolic membrane potential (Em) reducing the driving force for the NCX forward mode (Em-ENa/Ca) plasmic reticulum Ca2+-ATPase (SERCA) which mediates the re-uptake of Ca2+ ions into the sarcoplasmic reticulum while NCX mediates Ca2+ extrusion to the extracellular compartment. In higher mammals, the competition between SERCA and NCX shifts in favor of NCX, although SERCA still removes the major proportion of cytosolic Ca2+  Bers, . Moreove2+ transient amplitude in response to SEA0400. In OE 1 μM SEA0400 reduced the NCX-mediated Ca2+ removal rate on average to a level ranging between the basal hetKO rate and the rate of both wild-types  in a rabbit model of heart failure physiology, since there are substantial differences in cardiac CaNB, JS, SK, CP, and FM designed the study. NB, JS, SK, BB, and PP acquired the data. NB, JS, SK, BB, and DD performed the statistical analysis. All authors contributed substantially to the interpretation of the data. NB, JS, and SK wrote the manuscript. BB, PP, DD, GF, JG, UK, LE, CP, and FM critically revised the manuscript. All authors approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Intracellular Ca2+ concentrations are tightly maintained in the nanomolar range. Molecular mechanisms of Ca2+ uptake in bacteria remain elusive. Here we show that CtpE is responsible for Ca2+ uptake in Mycobacterium smegmatis. It represents a previously uncharacterized P-type ATPase family in bacteria. Disruption of ctpE in M. smegmatis resulted in a mutant with impaired growth under Ca2+-deficient conditions. The growth defect of the mutant could be rescued by Ca2+ or by ectopic expression of ctpE from M. smegmatis or the orthologous gene (Rv0908) from Mycobacterium tuberculosis H37Rv. Radioactive transport assays revealed that CtpE is a Ca2+-specific transporter. Ca2+ deficiency increased expression of ctpE, resulting in increased 45Ca2+ accumulation in cells. ctpE is a gene that is part of an operon, which is negatively regulated by Ca2+. The ctpE mutant also showed hypersensitivity to polymyxin B, increased biofilm formation, and higher cell aggregation, indicating cell envelope defects. Our work establishes, for the first time, the presence of Ca2+ uptake pumps of the energy-dependent P-type ATPase superfamily in bacteria and also implicates that intracellular Ca2+ is essential for growth and cell envelope integrity in M. smegmatis.Ca 2+ is essential for gene regulation, enzymatic activity, and maintenance of structural integrity of cell walls in bacteria. Bacteria maintain intracellular calcium concentrations in a narrow range, creating a gradient with low cytoplasmic calcium concentration and high extracellular calcium concentration. Due to this steep gradient, active pumps belonging to family 2 of P-type ATPases and antiporters are used for Ca2+ efflux, whereas Ca2+ uptake is usually carried out by channels. Molecular mechanisms of Ca2+ uptake in bacteria are still elusive and are mainly limited to a nonproteinaceous channel in Escherichia coli and a pH-dependent channel protein from Bacillus subtilis. Energy-dependent active transporters are not reported for Ca2+ uptake from any organism. Here we show that CtpE belonging to a family of previously uncharacterized bacterial P-type ATPases is involved in specific uptake of Ca2+ into Mycobacterium smegmatis. We also demonstrate that intracellular Ca2+ obtained through CtpE is essential for growth and maintenance of cell surface properties under Ca2+-deficient conditions.Ca These functions have been well characterized for eukaryotes and have begun to be characterized in prokaryotes  and 2 cytoplasmic loops after TM2 and TM4  broth and Middlebrook 7H9 medium, but growth of the mutant was slow in Sauton’s medium, and culturing the mutant in Sauton’s medium could not achieve a final cell density similar to that of the wild type (WT) (routine observation). Most P-type ATPases are cation transporters; we screened strain MHK1 against increasing concentrations of various cations and chelators. The MHK1 strain was sensitive to Ca2+-specific chelators EGTA and 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) compared to the WT ethane-1,2-diamine (TPEN), or phenanthroline  and the mutant (MHK1) in the presence of various metal ion chelators. Strains were grown in Luria-Bertani broth, and cell growth was monitored after 48 h of incubation at 37°C by measuring absorbance at 600 nm. (A) EDTA; (B) TPEN; (C) phenanthroline. Download FIG S2, TIF file, 4.6 MB.Growth of Copyright © 2017 Gupta et al.2017Gupta et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the ctpE, we complemented MHK1 with ectopic expression of ctpE under the control of the hsp60 promoter. Complemented strain MHK1 carrying pRHK2 was completely restored for the EGTA- or BAPTA-sensitive phenotype  did not compete for 45Ca2+ uptake, indicating that CtpE is a Ca2+-specific P-type ATPase.Cation selectivity of CtpE was examined by evaluating other cations as competitive inhibitors of Cahe cells . EquimolctpE revealed a putative operon with five upstream genes  and two downstream genes  in M. smegmatis . Amplification of each junction between the genes confirmed that expression of these genes is transcriptionally coupled   . No suchpromoter , indicatdditives  and D, i2+-deficient medium. CtpE was found to transport Ca2+ in M. smegmatis and was part of an operon carrying genes with putative function in cell envelope biosynthesis or modification. This demanded an evaluation of the cell envelope defects in the MHK1 strain. We checked sensitivity and survival of MHK1 under various stress conditions to evaluate cell envelope defects and observed that the mutant did not differ significantly from the wild type in the presence of SDS, lysozyme, or cell wall-inhibiting antibiotics such as ethambutol or isoniazid (2+ deficiency (1.0 mM EGTA) and were alleviated in the presence of excess Ca2+ (500 µM to 1.0 mM) in the growth medium. The phenotype of the complemented strain was similar to that of the wild type, indicating that CtpE-mediated Ca2+ uptake is important for the integrity of the cell envelope of M. smegmatis under Ca2+-deprived conditions.We routinely observed that the MHK1 colonies were dry and rough in comparison to relatively oily and smooth colonies of the wild type on Casoniazid . Interessoniazid  and alsosoniazid  and incrsoniazid . These d10.1128/mBio.01388-17.5FIG S4 FIG S4, TIF file, 8.4 MB.Survival and antibiotic sensitivity assays to monitor cell surface defects in the MHK1 mutant. Cultures were grown to mid-log phase in Sauton’s medium containing 1.0 mM EGTA. (A and B) The cells were harvested and inoculated in fresh medium containing SDS (0.05% [wt/vol]) (A) and lysozyme (250 µg/ml) (B); cell survival was monitored every 2 h by plating the samples on Sauton’s medium agar plates and counting the colony-forming units. The results are expressed as percentage survival with respect to the cell count at time zero. (C and D) Antibiotic sensitivity of the cultures was tested in Middlebrook 7H9 medium containing 0.05% Tween 80 and different concentrations of isoniazid (C) or ethambutol (D). Cell growth was monitored after 72-h incubation at 37°C by measuring absorbance at 600 nm. Download Copyright © 2017 Gupta et al.2017Gupta et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 2+ uptake transporter in M. smegmatis. To the best of our knowledge, this is the first Ca2+ uptake transporter belonging to the energy-dependent P-type ATPase superfamily reported from any organism. Distribution of CtpE orthologs in several bacterial divisions further indicates the importance and essential role of Ca2+ in the growth and physiology of prokaryotes. Orthologs of CtpE were identified from 290 strains and were absent in archaea and many other divisions in bacteria, indicating that this family of P-type ATPases has recently evolved for specific Ca2+ uptake required for essential physiological functions or survival of these species under low-Ca2+ growth conditions. We observed that strain MHK1 showed significant growth defect only in the presence of high concentrations of EGTA or BAPTA, which indicates that CtpE is required for acquisition of Ca2+ under Ca2+-deficient conditions and that M. smegmatis possesses yet uncharacterized alternate Ca2+ uptake mechanism(s) operating at higher Ca2+ concentrations.In this work, we demonstrate that CtpE is a CactpE is negatively regulated at the transcription level by Ca2+. This Ca2+-mediated regulation of ctpE may be due to a Ca2+-sensing/regulatory system in M. smegmatis. Eukaryotic calcium channels, plasma membrane calcium transporter (PMCA) and sarcoendoplasmic reticulum calcium transporter (SERCA) are known to be regulated at the transcription level by Ca2+-dependent signaling involving calmodulin and protein kinases  with functional similarity to eukaryotic calmodulin  was also purified and characterized from M. smegmatis (M. smegmatis remain unexplored. The presence of components of Ca2+-sensing (CAMLP) and regulatory (Cam-kinase) machinery and our observation of Ca2+-dependent regulation of ctpE in M. smegmatis tempt us to speculate on possible roles of CAMLP and Cam-kinase in ctpE regulation.We observed that  kinases . There akaryotes , but meclmodulin . This prlmodulin , and CAMnfection . A. Ca2+megmatis , but dirctpE is part of an operon with genes predicted to be involved in membrane/cell wall biosynthesis or integrity in M. smegmatis and other mycobacterial species  and penicillin-binding protein (PBP2) form a complex in the presence of Ca2+ through an EF-hand motif of PBP2. This complex is required for peptidoglycan polymerization and integrity of the cell envelope  possessed conserved DXDXDG motif known for Ca2+ binding  broth and LB agar supplemented with appropriate antibiotics. Mycobacterium smegmatis mc2155 (wild-type [WT]) and mutant strains were grown on LB medium supplemented with 0.05% Tween 80 or Middlebrook 7H9 medium supplemented with albumin-dextrose-catalase (ADC) or oleic acid-albumin-dextrose-catalase (OADC) or Sauton’s medium (pH 7.5) supplemented with 0.05% Tween 80 as described below for the particular experimental procedure.Bacterial strains and plasmids used in this study are listed in 10.1128/mBio.01388-17.7TABLE S2 TABLE S2, DOCX file, 0.02 MB.Primers used in this study. Download Copyright © 2017 Gupta et al.2017Gupta et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the M. smegmatis and orthologous sequences from other species was done by ClustalW, and a phylogenetic tree was constructed by neighbor-joining method using MEGA version 6.0  and its homologue Rv0908 from M. tuberculosis (using primers RVEF and RVER) were amplified, appropriately digested, and ligated into pSMT3 at the EcoRV or HindIII site to generate pRHK2 and pRHK3, respectively.DNA manipulations, such as PCR, restriction digestions, and ligations, were conducted using standard procedures . To genectpE in M. smegmatis mc2155 was generated by homologous recombination with sucrose counterselection  was used as a probe for confirmation of ctpE disruption in the MHK1 mutant. The labeling of the probe and hybridization and detection of hybridizing fragment were carried out according to the manufacturer’s instructions .Five micrograms of the genomic DNA was restriction digested with SacI, and Southern hybridization was performed as described previously . The kanM. smegmatis mc2155 and MHK1 were grown in Sauton’s medium supplemented with 0.05% Tween 80 for 24 h, diluted 1:100, and added to medium containing 1.0 mM EGTA. Restoration of growth with different cations was monitored with the addition of 0.1 to 1.0 mM concentrations of CaCl2, 1.0 mM MgCl2, BaCl2, MnCl2, NiCl2, or CuSO4, or 0.5 mM CoCl2 or ZnCl2. Cells were cultured for 48 h with shaking at 37°C, and growth was monitored at 600 nm.To check the growth of strain MHK1 with different cations, starter cultures of M. smegmatis MHK1 and mc2155 were transformed with pSMT3, pRHK2, or pRHK3 by electroporation. Complementation was checked by growing all strains in Sauton’s medium supplemented with 0.05% Tween 80 and 50 µg ml−1 hygromycin with different concentrations of EGTA or 1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) for 48 h, and the growth was monitored at 600 nm.M. smegmatis strains were grown in Sauton’s medium without any additive or with 1.0 mM EGTA or 1.0 mM Ca2+. The cells were harvested, washed twice, and resuspended at a cell density of 1.0 OD600  unit/ml in Sauton’s medium without any additive. The cells were incubated at 37°C for 15 min, and then 45Ca2+ uptake was initiated by the addition of 45Ca2+ to a final concentration of 1.0 μCi/ml and 10 mM cold carrier CaCl2. At the indicated time, 1.0-ml aliquots were filtered through 0.45-μm nitrocellulose filters, which were prewashed with 1.0 mM EGTA containing Hanks’ balanced salt solution (HBSS). The cells were washed twice with 1.0 mM EGTA containing HBSS, dried in an oven, and immersed in 1.0 ml of toluene-based scintillation fluid, and the amount of cell-accumulated 45Ca2+ was determined by liquid scintillation counting using a Wallac MicroBeta TriLux instrument (Perkin Elmer). Identical assay reaction mixtures were incubating on ice as controls. The counts from the ice controls were deducted from the counts of the test reaction mixtures incubated at 37°C.M. smegmatis mc2155 was isolated using an RNA isolation kit (Qiagen). The RNA was treated with DNase I and purified using an RNeasy kit (Qiagen). The absence of DNA contamination in RNA samples was confirmed by the absence of PCR amplification of sigA using SIGAF and SIGAR primers set by Taq polymerase.Total RNA from ctpE operon, approximately 400- to 600-bp junction regions between two genes were amplified with the help of junction primers listed in sigA was amplified as a positive control of RT-PCR using SIGAF and SIGAR primers.To characterize the M. smegmatis mc2155 cultures were grown in Sauton’s medium until they reached an OD600 of 1.0. The cultures were then treated with 7.5 mM EGTA or 2.5 mM CaCl2 and allowed to grow for 2 h, and the total RNA was isolated using RNA isolation kit (Qiagen). Total RNA was quantified with a NanoDrop spectrophotometer . Semiquantitative RT-PCR is carried out using 1.0 μg total RNA of each sample with a One-Step RT-PCR kit (Qiagen). The reaction mixture was incubated at 50°C for 30 min, followed by denaturation at 95°C for 15 min. The products were then amplified by 30 cycles, with 1 cycle consisting of denaturation at 94°C for 30 s, annealing  at 60°C for 1 min, and extension at 72°C for 30 s. The final extension was carried out at 72°C for 10 min. Amplification products were detected in 1.5% agarose gels along with a 100-bp ladder. sigA was used as an internal control and was amplified using sigsA2F and -A2R primers. Quantitation of amplified PCR product was done by densitometry using AlphaEaseFC 4.0. Densitometry values of ctpE and echA were normalized with internal control sigA. P values were calculated using GraphPad and indicated as follows: *, P < 0.05; **, P < 0.01. Experiments were performed three times independently.To perform semiquantitative RT-PCR, the M. smegmatis strains in Middlebrook 7H9 medium supplemented with ADC for 24 h and then inoculating the cultures as 1:200 dilutions in fresh medium containing a range of antibiotic concentrations, followed by incubation at 37°C and 200 rpm for 72 h. Growth was monitored at 600 nm.The antibiotic sensitivity assays were performed by growing 2 or 1.0 mM EGTA were inoculated with 40 µl of mid-log-phase culture of each strain. Plates were sealed with Parafilm and incubated undisturbed at 37°C, and biofilm formation was observed after 5 days.Biofilm formation was analyzed by the method of Ojha et al.  with smaAggregation assays were performed by the method of Klepp et al.  with sli


We reported previously that these Ca2+ signals, which arise from Ca2+ entry and subsequent amplification by Ca2+ release through ryanodine receptor (RyR) channels, promote mitochondrial network fragmentation and reduce RyR2 expression. Here, we examined if AβOs, by inducing redox sensitive RyR-mediated Ca2+ release, stimulate mitochondrial Ca2+-uptake, ROS generation and mitochondrial fragmentation, and also investigated the effects of the antioxidant N-acetyl cysteine (NAC) and the mitochondrial antioxidant EUK-134 on AβOs-induced mitochondrial dysfunction. In addition, we studied the contribution of the RyR2 isoform to AβOs-induced Ca2+ release, mitochondrial Ca2+ uptake and fragmentation. We show here that inhibition of NADPH oxidase type-2 prevented the emergence of RyR-mediated cytoplasmic Ca2+ signals induced by AβOs in primary hippocampal neurons. Treatment with AβOs promoted mitochondrial Ca2+ uptake and increased mitochondrial superoxide and hydrogen peroxide levels; ryanodine, at concentrations that suppress RyR activity, prevented these responses. The antioxidants NAC and EUK-134 impeded the mitochondrial ROS increase induced by AβOs. Additionally, EUK-134 prevented the mitochondrial fragmentation induced by AβOs, as previously reported for NAC and ryanodine. These findings show that both antioxidants, NAC and EUK-134, prevented the Ca2+-mediated noxious effects of AβOs on mitochondrial function. Our results also indicate that Ca2+ release mediated by the RyR2 isoform causes the deleterious effects of AβOs on mitochondrial function. Knockdown of RyR2 with antisense oligonucleotides reduced by about 50% RyR2 mRNA and protein levels in primary hippocampal neurons, decreased by 40% Ca2+ release induced by the RyR agonist 4-chloro-m-cresol, and significantly reduced the cytoplasmic and mitochondrial Ca2+ signals and the mitochondrial fragmentation induced by AβOs. Based on our results, we propose that AβOs-induced Ca2+ entry and ROS generation jointly stimulate RyR2 activity, causing mitochondrial Ca2+ overload and fragmentation in a feed forward injurious cycle. The present novel findings highlight the specific participation of RyR2-mediated Ca2+ release on AβOs-induced mitochondrial malfunction.Amyloid β peptide oligomers (AβOs), toxic aggregates with pivotal roles in Alzheimer’s disease, trigger persistent and low magnitude Ca Familiachannels . Of notelum (ER) . Further+ levels .2+ signals in primary hippocampal neurons; these signals arise initially from Ca2+ entry through N-Methyl-D-aspartate (NMDA) receptors and are subsequently amplified via RyR channels co-stimulated by Ca2+ entry signals and the increased ROS levels produced by AβOs  induce anomalous Ca by AβOs . Further by AβOs , are 20% by AβOs . Interes neurons . Moreove neurons , whereas neurons  and spat neurons  increase2+ signals elicited by AβOs prevent the spine remodeling prompted by BDNF, and provoke mitochondrial network fragmentation  induces mitochondrial fragmentation in neurons , a physiological precursor of cellular glutathione (GSH) synthesis, prevents the mitochondrial network fragmentation and RyR2 knockdown mediated by RyR channel activation in response to AβOs  was from Bachem Inc. . Fluo4-AM, MitoSOXTM Red Mitochondrial Superoxide Indicator, MitoTracker® Orange CMTMRos, anti-rabbit Alexa Fluor® 488 and anti-mouse Alexa Fluor® 635 were from Molecular Probes, Inc. . Hexafluoro-2-propanol (HFIP) and CMC were from Merck , Neurobasal and Dulbecco’s modified essential medium (DMEM), B27 supplement and lipofectamine 2000 were from Gibco . DOTAP Liposomal Transfection Reagent was from Sigma–Aldrich . Phosphodiester oligonucleotides (ODNs) were from Integrated DNA Technologies . The mito-Pericam plasmid was donated by Dr. V. Eisner. Bicinchoninic acid assay (BCA) kit and mHsp-70 antibody were from Pierce Biotechnology . Ryanodine was from Alexis . PDVF membranes were from Millipore . RyR2 antibody and Rhod2-AM was from Thermo-Fisher . Gp91 ds-tat was from AnaSpec .Aβ peptide . Supernatants with soluble AβOs were transferred to sterile tubes and protein levels were determined with a BCA kit. Fresh preparations of AβOs were used in all experiments.The AβFIP film . This fiin vitro (DIV) in a humidified 5% CO2 atmosphere at 37°C prior to experimental handlings. Mature hippocampal cultures were enriched in neurons with a glial content <24% . Cells were rinsed three times with PBS, and coverslips were mounted in DAKO mounting medium for morpho-topological analysis of the mitochondrial network. Quantification of the percentage of neurons with fragmented mitochondria was carried out as described previously  for 10 min, rinsed three times with PBS, incubated with 10% normal goat serum plus 0.1% Triton X-100 (blocking-permeant solution) for 1 h and then immunolabeled by overnight incubation at 4°C with mHsp-70 diluted in blocking solution (1/750). After this incubation period, cultures were rinsed three times with PBS and were incubated for 1 h at room temperature with Alexa Fluoreviously , 2014. TNeurons (14–21 DIV) were treated with 500 nM AβOs at the microscope stage, or for different incubation periods in the culture plates, depending on the type of experiment performed.∗T∗C GCCCGCATCAGCC∗A∗T-3′; ODN Scrambled (ODN Scr), 5′-C∗G∗GCAGGAGTCTGTG C∗G∗C-3. The ODN Scr was used as control, as previously described  with the following sequences. ODN RyR2: 5′-Tescribed . LiposomRyR2 mRNA levels were determined by RT-PCR performed in a MX3000P Stratagene amplification system  using the DNA binding dye SYBR green and the following previously described Primer sense/Primer antisense sequences: 5′-AATCSanMartínGTGGCGGAATTTCTTG-3′/5′-TCTCCCTCAGCCTTCTCCGGTTC-3′ . Levels 2, 1 MgCl2, 25 HEPES-Tris, pH 7.3) for 30 min at 37°C. After washing three times with Tyrode, 500 nM AβOs were added to the cultures at the microscope stage and fluorescence images of intracellular Ca2+ signals were obtained every 15 s in an inverted confocal microscope  or every 3 s in an inverted confocal microscope (Nikon C2+). Regions of interest (ROIs) were determined in cell bodies and neurites. Relative Ca2+ levels are presented as F/F0 values, where F0 corresponds to the basal fluorescence and F to the experimental fluorescence. Experiments were done at room temperature (20–22°C).Cells were preloaded with 5 μM Fluo4-AM in Tyrode solution  were recorded every 3 s in an Olympus Disk Scanning Unit (DSU) IX 81 confocal microscope  using 60× oil immersion objective, excitation 420 nm and Hg/Ar lamp. Changes in Ca2+ levels are presented as F/F0 values, where F corresponds to the experimental fluorescence and F0 to the basal fluorescence. Experiments were done at room temperature (20–22°C).Neurons at 14 or 15 DIV were transiently transfected with the mito-Pericam plasmid using a ratio of 1:3 DNA:lipofectamine 2000 as previously described . Twenty-2+ signals were obtained every 3 s in an inverted confocal microscope (Nikon C2+). ROIs were determined in cell bodies and neurites. Relative Ca2+ levels are presented as F/F0 values, where F0 corresponds to the basal fluorescence and F to the experimental fluorescence. Experiments were done at room temperature (20–22°C).Neurons at 14–21 DIV were incubated with 2.5 μM Rhod2 for 30 min, washed three times with Tyrode solution and incubated for additional 30 min to allow mitochondrial loading with Rhod2. Next, cells were transferred to Tyrode solution containing 5 μM Fluo4 and incubated for an additional 30 min period. Cells were then rinsed three times with Tyrode and AβOs (500 nM) were added to the cultures at the microscope stage. Simultaneous fluorescence images of intracellular and mitochondrial CaF/F0 values, where F0 corresponds to the basal fluorescence levels and F to the experimental fluorescence. Experiments were performed at room temperature (20–22°C).Cultures were treated for 1 h with 50 μM ryanodine, for 30 min with 10 mM NAC, or for 2 h with 20 μM EUK-134, in Neurobasal medium supplemented with B-27. Cultures were then placed in modified Tyrode solution for subsequent loading with 1 μM MitoSOX for 20 min at 37°C. After washing three times with modified Tyrode solution, AβOs (500 nM) were added to the cultures at the microscope stage. The fluorescence images generated by the mitochondrial superoxide probe in primary hippocampal neurons (identified as such by morphology) were recorded every 5 s in a confocal microscope . Fluorescence signals are presented as F/F0 values, where F0 corresponds to the basal fluorescence and F to the experimental fluorescence. Experiments were performed at room temperature (20–22°C).Cultures at 14 or 15 DIV were transfected transiently with the HyperMito plasmid  at a ratio of 1:3 DNA:lipofectamine 2000. Twenty-four hours after transfection, cultures were treated for 1 h with 50 μM ryanodine, for 30 min with 10 mM NAC, for 2 h with 20 μM EUK-134, or with vehicle in Neurobasal plus B27 medium. After three rinses with Tyrode solution, 500 nM AβOs were added to the cultures at the microscope stage. The fluorescent signals generated by the mitochondrial hydrogen peroxide probe were recorded from neuronal cells (identified as such by morphology) every 3 s in a confocal microscope . Relative mitochondrial hydrogen peroxide levels are presented as Mitochondria were identified by staining fixed cultures with mHsp-70, as we previously described , 2014. Tz-axis. Based on their volumes, we defined four different clusters to characterize mitochondrial connectivity as previously described . The mean volume of single mitochondria was used to define connected clusters: (i) 1–3 mitochondria (0–0.45 μm3); (ii) 4–10 mitochondria (0.45–1.5 μm3); (iii) 11–50 mitochondria (1.5–7.5 μm3); (iv) over 50 mitochondria (>7.5 μm3). Values obtained with control neurons were compared to those obtained from neurons treated with 500 nM or 1 μM AβOs for 24 h.3D models were reconstructed from successive xy-images along the escribed . First, t-test or one-way ANOVA followed by Bonferroni’s post hoc test.The significance of differences in the experiments was determined using paired Student’s 2+ entry signals via NMDA receptors, which promote RyR-mediated Ca2+-induced Ca2+ release , displayed similar Ca2+ signal generation in response to AβOs as controls. Based on these findings, we suggest that AβOs stimulate NOX2 activity, presumably via NMDA receptor stimulation  or with the RyR channel agonist 4-CMC (0.5 mM). Addition of AβOs  produced a significant decrease in mito-Pericam fluorescence compared to that registered under basal levels , indicating that AβOs induce mitochondrial Ca2+ entry. Quantification of fluorescence changes revealed that, within 1 min after AβOs or 4-CMC addition, neurons displayed significantly lower fluorescence relative to neurons treated with vehicle ; after 500 s, the decrease was significantly higher in 4-CMC-treated compared to AβOs-treated neurons.Mito-Pericam is a plasmid that expresses a Ca binding , which i2+ release from the ER underlies the AβOs-induced mitochondrial Ca2+ increase, we pre-incubated neurons for 1 h with 50 μM ryanodine (Rya), which in these conditions abolishes RyR-mediated Ca2+ release without causing Ca2+ depletion from the ER . Previous work indicated that AβOs promote cytoplasmic ROS production , comprised of a hydroethidine linked to a triphenylphosphonium cationic group that target this probe to the mitochondrial matrix in response to the negative membrane potential , a RyR-channel agonist, exhibited an increase in MitoSOX fluorescence , which was significantly higher than the increase produced by AβOs. The addition of vehicle did not change probe fluorescence.To determine mitochondrial superoxide levels we used the MitoSOXotential . OxidatiTM-Mito plasmid. This plasmid codes for the mitochondrial protein HyPer-mito that has a circularly permuted yellow fluorescent protein inserted into the regulatory domain of the prokaryotic hydrogen peroxide-sensing protein (OxyR) . In contrast, neurons in cultures pre-incubated for 1 h with 50 uM ryanodine to prevent RyR-mediated Ca2+ release did not exhibit changes in probe fluorescence in response to AβOs . The quantification of the fluorescence recorded 1200 s after AβOs addition is shown in Figure 2D. Altogether, the combined results illustrated in Figure 2 indicate that AβOs-induced RyR-mediated Ca2+ release has a key role in AβOs-induced mitochondrial superoxide and hydrogen peroxide generation.To detect mitochondrial hydrogen peroxide generation, primary hippocampal cultures were transiently transfected with the Hypern (OxyR) , allowinFigure 3A shows representative fluorescence images of mitochondrial superoxide generation recorded before (left) and 1500 s (right) after AβOs addition, in the presence or absence of NAC. As illustrated in these images, NAC prevented the mitochondrial superoxide increase elicited by AβOs. The quantification of the results from several experiments indicates that NAC completely prevented the superoxide increase in the mitochondria . In agreement with these findings, cultures pre-incubated for 2 h with the mito-protector agent EUK-134 (20 μM) exhibited a significant decrease in neuronal superoxide levels following AβOs addition, as observed in the pseudo color images shown in Figure 3D. Figure 3E illustrates the quantification of the kinetics of the MitoSox fluorescence changes and Figure 3F, the endpoint fluorescence values.We evaluated the effects of the general antioxidant NAC on the mitochondrial superoxide production induced by AβOs. For this purpose, neurons were pre-incubated for 1 h with NAC (10 mM), and then AβOs were added at the microscope stage. Figure 4A shows representative fluorescence images of neuronal mitochondrial H2O2 generation before (left) and 1200 s (right) after AβOs addition, in the presence or absence of NAC  or EUK-134 . Both NAC and EUK-134 prevented the increase in mitochondrial H2O2 levels caused by AβOs addition. Quantification of the kinetics of hydrogen peroxide generation is shown in Figures 4B,E, while the fluorescence intensities measured at the endpoint of the experiments are shown in Figures 4C,F.The effects of NAC and EUK-134 on AβOs-induced mitochondrial hydrogen peroxide production were tested next. For this purpose, cultures were pre-incubated with NAC and EUK-134 as described above. Based on these combined findings, we conclude that both antioxidants, NAC and EUK-134, prevent the increases in mitochondrial superoxide and hydrogen peroxide levels induced by AβOs.2+ release (3) was used to define all clusters . As previously described  displaying a characteristic organization of their mitochondrial network, which may reflect specific cellular demands in the neuronal soma  and neurites . Compared to a representative control neuron , the continuity of the mitochondrial network of a neuron incubated for 24 h with 500 nM AβOs exhibited a loss, and the proportion of small mitochondria in soma and neurites increased . The quantification of morpho-topological analysis of mitochondrial fragmentation revealed that AβOs decreased the fraction of the biggest clusters (>7.5 μm3) and increased the proportion of the intermediate clusters of mitochondria (1.5–7.5 μm3), in the soma  as well as in the neurites . This effect was dose-dependent, but we set the subsequent experiments with the lower concentration of AβOs, 500 nM, which we have reported to be sub-lethal . In contrast, a significantly higher percentage of neurons (67%) treated with 500 nM AβOs for 24 h contained punctuate mitochondria, exposing noteworthy fragmentation of the mitochondrial network . Incubation with 20 μM EUK-134 before AβOs treatment significantly decreased  the fraction of neurons exhibiting fragmented mitochondria; 20 μM EUK-134 by itself  did not elicit significant changes in the content of fragmented mitochondrial (11%) when compared to the controls . Theses results indicate that the EUK-134 mitochondrial antioxidant prevents mitochondrial fragmentation induced by AβOs.We investigated next the effects of the mitochondrial antioxidant EUK-134 on the mitochondrial fragmentation induced by AβOs. Analysis of fixed control neurons stained with MitoTracker Orange revealed that only 5% of primary hippocampal neurons contained fragmented mitochondria, while most of the neurons exhibited filamentous mitochondria in neurites and soma  and protein contents , determined in homogenates of the whole primary culture. We studied next the impact of RyR2 knockdown on agonist-induced RyR-mediated cytoplasmic Ca2+ signals elicited by 4-CMC, and found that neurons in ODN RyR2 transfected cultures exhibited 40% lower Ca2+ signals when compared to neurons present in cultures transfected with the scrambled oligonucleotide (ODN Scr) .Oligotransfection of primary hippocampal cultures with an oligodeoxynucleotide against RyR2 (ODN RyR2) reduced by 50% RyR2 mRNA . As reported previously . The fluorescence intensities observed in ODN RyR2 and ODN Scr-treated neurons revealed that ODN RyR2 transfection caused a significant reduction in both the cytoplasmic  and the mitochondrial  Ca2+ signals induced by AβOs. The quantification of the last fifteen seconds of the average of three experiments shows that these differences are statistically significant .To evaluate if RyR2 knockdown affected AβOs-induced cytoplasmic and mitochondrial Caeviously , we confFigures 8A,B). Transfection with ODN RyR2 markedly reduced almost to zero the percentage of neurons that exhibited a mitochondrial punctuate pattern, even after AβOs treatment . Based on these results, we propose that Ca2+ release mediated by the RyR2 isoform plays a central role in AβOs-induced mitochondrial fragmentation.We further investigated the effects of transfection with ODN RyR2 on AβOs-induced mitochondrial fragmentation. Analysis of control neurons transfected with ODN Scr and loaded with MitoTracker Orange, revealed that about 15% of primary hippocampal neurons contained fragmented mitochondria, whereas 24 h after 500 nM AβOs addition 53% exhibited punctuate mitochondria, revealing fragmentation of the mitochondrial network .Due to the activity of the electron transport chain, mitochondria are the major sources of superoxide and hydrogen peroxide production in cells even under physiological conditions . An incrA previous report showed that neurons treated for 1 h with 500 nM AβOs display increased mitochondrial superoxide generation, measured with the MitoSOX probe . Here, wUncontrolled mitochondrial ROS generation may interfere with the morphology of the mitochondrial structure. The energetic requirements of a cell are related to its function and to the number of mitochondria, their morphology and distribution in the cytoplasm, which is particular to each type of cell . In the The first evidence linking AD with modifications in the structure of the mitochondrial network was reported in fibroblasts from AD patients, which exhibit increased fused mitochondria presumably caused by a decrease in the expression of the fission protein Drp-1 . Subsequ2+ release from the ER. As a result, mitochondria would fail to take up Ca2+, preventing the increased ROS production caused by Ca2+ uptake. Previous reports indicate that NAC protects the hippocampus from oxidative stress, apoptosis, and Ca2+ entry  for 24 h, increased the population of mitochondria with volumes <7.5 μm2+ transfer from the ER to the mitochondria induced by acute treatment with AβOs, and prevents the ensuing disruption of the mitochondrial network. Based on these results, we propose that the initial RyR2 down-regulation induced by AβOs represents an early protective neuronal response from the RyR2-mediated noxious effects of AβOs on mitochondrial function, which presumably contribute to AβOs-induced early synaptotoxicity. This proposal agrees with previous findings showing that ryanodine, at inhibitory concentrations, prevents the mitochondrial fragmentation induced by acute AβOs treatment (We describe here novel findings highlighting the key role of the RyR2 isoform in the mitochondrial dysfunctions induced by acute AβOs treatment. We showed previously that RyR2 up-regulation accompanies the increase in spine density induced by BDNF; RyR2 up-regulation also occurs following high frequency field stimulation of primary hippocampal cultures and spatial memory training . Conversreatment . In addireatment . TherefoFigure 9. PL performed the analysis of some experiments, and contributed to the writing of the manuscript. BB was the responsible for Abeta oligomers preparations. JG was in charge of the primary hippocampal cultures. AG and SH provided support for microscopy image analysis. CH and AP-L participated in the experimental design, in the interpretation of the results, in manuscript writing and also provided most of the financial support for the work.CS and PV performed most of the experimental work and analysis. TA contributed with the experimental design, performed some of the experiments and generated the final scheme presented as The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Control of calcium binding to and dissociation from cardiac troponin C (TnC) is essential to healthy cardiac muscle contraction/relaxation. There are numerous aberrant post-translational modifications and mutations within a plethora of contractile, and even non-contractile, proteins that appear to imbalance this delicate relationship. The direction and extent of the resulting change in calcium sensitivity is thought to drive the heart toward one type of disease or another. There are a number of molecular mechanisms that may be responsible for the altered calcium binding properties of TnC, potentially the most significant being the ability of the regulatory domain of TnC to bind the switch peptide region of TnI. Considering TnI is essentially tethered to TnC and cannot diffuse away in the absence of calcium, we suggest that the apparent calcium binding properties of TnC are highly dependent upon an “effective concentration” of TnI available to bind TnC. Based on our previous work, TnI peptide binding studies and the calcium binding properties of chimeric TnC-TnI fusion constructs, and building upon the concept of effective concentration, we have developed a mathematical model that can simulate the steady-state and kinetic calcium binding properties of a wide assortment of disease-related and post-translational protein modifications in the isolated troponin complex and reconstituted thin filament. We predict that several TnI and TnT modifications do not alter any of the intrinsic calcium or TnI binding constants of TnC, but rather alter the ability of TnC to “find” TnI in the presence of calcium. These studies demonstrate the apparent consequences of the effective TnI concentration in modulating the calcium binding properties of TnC. Further complicating molecular insight, the disease-associated protein modifications can also impact the myocyte's innate ability to tune Ca2+ sensitivity via phosphorylation, so that only under certain conditions might the apparent Ca2+ sensitivity even appear altered  why the Tn complex has such high apparent Ca2+ sensitivity and slow Ca dissociation (compared to the isolated protein) that is drastically reduced and accelerated, respectively, when the Tn complex is incorporated onto the thin filament; (2) why a large proportion of Tn modifications seem to have no effect on the apparent Ca2+ binding properties of the isolated Tn complex, yet differences emerge when placed in the context of the thin filament; (3) that there are several different molecular mechanisms within and outside of the Tn complex that influence the intrinsic and/or apparent Ca2+ binding properties of TnC; and (4) at least four states of TnC are required to simulate the apparent Ca2+ binding properties of TnC in different experimental and diseased conditions.With respect to TnI availability, there are fascinating phenomena that occur at the boundaries of how we think about the mechanical and chemical world. One such behavior, effective concentration, emerges when reactants are restricted to interact in very confined spaces, as occurs when two reactants are physically tethered together . We generated two TnC-TnI chimeras consisting of the N-terminal domain of human cardiac TnC (residues 1–89) with the C-terminal domain of human cardiac TnI (residues 128–211) connected by a flexible and cleavable linker containing a site for the Tobacco Etch Virus protease, which contained the sequence GGAGGENLYFQG. For the F27W chimera, the endogenous Cys residues within TnC were converted into Ser and Phe 27 was converted to Trp, resulting in the following protein sequence: MDDIYKAAVEQLTEEQKNEFKAAFDIWVLGAEDGSISTKELGKVMRMLGQNPTPEELQEMIDEVDEDGSGTVDFDEFLVMMVRSMKDDSGGAGGENLYFQGLTQKIFDLRGKFKRPTLRRVRISADAMMQALLGARAKESLDLRAHLKQVKKEDTEKENREVGDWRKNIDALSGMEGRKKKFES. For the T53C-IAANS chimera, the endogenous Cys residues within TnC were converted into Ser, Thr 53 was converted to Cys (resulting in the following protein sequence: MDDIYKAAVEQLTEEQKNEFKAAFDIFVLGAEDGSISTKELGKVMRMLGQNPCPEELQEMIDEVDEDGSGTVDFDEFLVMMVRSMKDDSGGAGGENLYFQGLTQKIFDLRGKFKRPTLRRVRISADAMMQALLGARAKESLDLRAHLKQVKKEDTEKENREVGDWRKNIDALSGMEGRKKKFES) and labeled with IAANS as previously described  and dialyzed at least four times against 1 L of the same buffer. The solution was then loaded onto an SQ-15 column equilibrated with buffer A. After an initial washing with Buffer A, a gradient was applied with 0–25% of buffer B (buffer A with 1 M NaCl). Fractions were collected and then dialyzed against 4 L of 10 mM MOPS, 150 mM KCl, at pH 7.0 at least four times.2 were added to 2 ml of titration buffer  with constant stirring. The [Ca2+] free was calculated using the computer program EGCA02 developed by Robertson and Potter as previously described . 0.5 μM human cardiac TnCF27W was titrated with Ca2+ in the absence or presence of up to 10 μM TnI128−180. The F27W chimera was also titrated with Ca2+ in the absence or presence of 3 mM Mg2+.All steady-state fluorescence measurements were performed using a Perkin-Elmer LS55 spectrofluorimeter at 15°C. Trp fluorescence was excited at 295 nm and monitored at 320 nm as microliter amounts of CaCl2+ dissociation rates were characterized using an Applied Photophysics model SX.20 stopped-flow instrument with a dead time of 1.4 ms at 15°C. IAANS fluorescence was excited at 330 nm with emission monitored through a 420–470 nm band-pass interference filter . Data traces  were fit with a single exponential equation to calculate the kinetic rates. The working buffer used for the kinetic measurements was 10 mM MOPS, 150 mM KCl, at pH 7.0. Ten millimeters EGTA was utilized to remove saturating Ca2+ from 1 μM of the human cardiac T53C-IAANS TnC (in the absence or presence of increasing concentrations of TnI128−180), uncleaved 0.5 μM T53C-IAANS chimera, or cleaved T53C-IAANS chimera (in the presence or absence of increasing concentrations of TnI128−180). The chimera was cleaved overnight at 4°C by the addition of one part TEV protease for every five parts of chimera in 10 mM MOPS, 150 mM KCl, at pH 7.0. Since we do not complex as much EGTA with Ca2+ as will occur during the titration experiments, we do not need to use as much MOPS to buffer the pH. Control experiments confirmed that using buffer containing 200 mM MOPS instead of 10 mM MOPS did not affect the apparent rate of Ca2+ dissociation from the Tn complex following T53C-IAANS TnC fluorescence (data not shown).Ca2+ dissociation and association rate studies, we normalized the change in the concentration of the species overtime and simply overlaid the simulations onto the actual experimental data.Using Scilab, an open source numerical computational package, we solved the differential equations to obtain the time-dependent concentrations of each species given a set of rate constants and initial concentrations (described below). For the two state simulations, we plotted the TnC-Ca species as the fluorescent state and for all other simulations we plotted the TnC-Ca-TnI species as the fluorescent state. For the steady-state and transient occupancy studies, the concentration of these species were subsequently converted to a percentage of the total TnC concentration and simply overlaid onto the actual experimental data. For the Ca−22 m3. By calculating the molarity of 1 TnI in this space using Avogradro's number we are able to estimate an effective concentration of ~13 mM. If we include more residues, 134–155, we estimate a lower limit of ~3 mM. This is very much in line with previous estimates of the effective concentration of a chimeric TnC-TnI protein  and assume it to be the radius of a sphere, we obtain a total volume of 1.25 × 102+ binding when Mg2+ was explicitly considered, we ran initial simulations with 1 μM TnC and 3 mM MgCl2 to equilibrium. This was then followed by running a loop where different levels of calcium from 0.0362 to 1000 μM were inputted and run to equilibrium. For the loop, each simulation was run to a time span of at least 0.5 s for each inputted Ca2+ concentration to reach equilibrium. A resulting plot of pCa vs. activated TnC ([TnC-Ca-TnI]) was developed.To simulate steady-state Ca2 and initially ran a simulation (at least 0.5 s time span) to determine the equilibrium concentrations of species resulting from Mg2+ binding. This was followed by inputting [Ca2+] of 200 μM. After running a simulation for at least 0.5 s, we inputted [EGTA] of 10 mM and ran a simulation. A plot of time vs. [TnC-Ca-TnI] was developed and outputted to a file.As with steady-state calcium binding, we initially began the simulation with 1 μM TnC and 3 mM MgCl2, and 600 μM EGTA. After running a simulation to equilibration, we inputted [Ca2+] levels of 12.5, 25, 50, and 1000 into the simulation. The simulations were normalized to the highest [Ca2+] level and a time vs. [TnC-Ca-TnI] plot was developed. We also performed studies without EGTA simulating the response of thin filaments to different [Ca2+] levels: 2.5–20 μM.We initially began the transient occupancy simulations with 1 μM TnC, 3 mM MgCl2+ binding properties of cardiac TnC vary substantially when studied in different systems  when only the isolated TnC is investigated, intermediate when the Tn complex is reconstituted onto the thin filament and highest in the isolated Tn complex or when the thin filament is bound by rigor myosin heads either in reconstituted thin filaments or Tn exchanged myofibrils. Similarly, the apparent rate of Ca2+ dissociation from TnC in these different systems somewhat scale proportionately to the change in apparent affinity . Likewise, Figure 2+ dissociation from TnC (T53C-IAANS) slows with increasing concentration of TnI128−180down to a limit. This limit too approaches the apparent Ca2+ dissociation rate from a chimeric protein in which the first 89 N-terminal residues of TnC (T53C-IAANS) were physically tethered by a short peptide linker to the C-terminal domain of human cardiac TnI  at a stoichiometric ratio of one to one.One of the obvious differences between the simplest systems is the presence or absence of TnI. It is well-documented that the binding of the C-terminal domain of TnI to the regulatory domain of TnC increases the apparent CaC Figure . As can 2+ dissociation is no longer observable  site that can be specifically cleaved by TEV Figure . As can 6 Å3. If we then place a single switch peptide into this volume we can calculate what the “effective concentration” of this peptide would be for a TnC that shares this volume space, ~3000 μM. This calculated value is similar to that estimated for another TnC-TnI chimeric protein by NMR  or a three state model  behind disease mutations, which are computationally forced to focus on very specific areas of interest, such as the precise coordination geometry of the Cat Figure . SimilarM Figure . Since Me Figure . In essed Figure . Thus, t2+ association and dissociation rates from TnC , it is reasonable to assume that it is necessary to at least have a three state model to predict TnC behavior in muscle. In fact, a three state model has been utilized quite successfully to simulate the apparent Ca2+ binding properties of cardiac myofibrils , whereas the Ca2+ dissociation rate approaches an asymptote of zero. Thus, a three state model that does not allow for Ca2+ to dissociate from TnC until TnI dissociates predicts that the apparent Ca2+ sensitivity would approach infinity and the Ca2+ dissociation rate would approach zero as the TnI concentration is continually increased. Clearly this is not the case based on our peptide and chimera studies demonstrated in Figure 2+-TnI complex can also dissociate by releasing Ca2+ as well  with an intrinsic Ca2+ association rate similar to that predicted by a two-state model for the isolated Tn complex .Thus, we suggest at minimum a four state model of TnC CaC Figure . Similar2+ binding properties of the isolated Tn complex. By only lowering the effective concentration of TnI to ~8 μM  our four state model also predicts the apparent Ca2+ binding properties of the reconstituted thin filament's: (1) steady-state Ca2+ binding affinity , Figures y Figure ; (2) Ca2e Figure ; (3) Ca2s Figure ; and (4)s Figure . Currents Figure , one cou2+ binding properties between different systems, we were curious if this mechanism might also be used to explain how certain disease associated mutations alter the apparent Ca2+ sensitivity of TnC. Excitingly, Figures 2+ binding properties of the thin filament without altering any intrinsic Ca2+ or TnI binding properties of TnC. However, the remaining Tn modifications we have studied require altering the intrinsic properties of TnC as well, in order to simulate the Ca2+ binding properties of the Tn complex and thin filament (manuscript in preparation). Collecting both steady-state and kinetic data from isolated Tn and the thin filament is essential to helping to elucidate whether a change in Ca2+ binding properties is due to intrinsic and/or extrinsic factors.Now that we have demonstrated that changing only the effective concentration of TnI connects TnC's Ca2+ binding as well as the speed of muscle mechanics is phosphorylation of cardiac TnI at serine residues 23 and 24  we are able to reconcile our experimental findings. Once we were able to correct for the Ca2+ buffering differences in our experimental systems, we were able to simulate the behavior of TnI S23/24D in both our data sets by making only a single intrinsic rate change of accelerating the TnI dissociation from TnC of 110–460/s (maintaining the effective concentration of TnI at 8 μM similar to the wild type condition).A major modulator of thin filament Cay Figure . However2+ binding properties and Ca2+ dissociation rate of the isolated Tn complex containing TnI S23/24D were nearly identical to that of the wild type Tn complex. This result can be simply modeled by again assuming the isolated troponin complex has a very high effective TnI concentration (>1000 μM in this case). Thus, a high effective TnI concentration can overcome the Ca2+ desensitizing effects of TnI S23/24D. On the other hand, TnI S150D, a phosphomimetic that models AMP kinase phosphorylation of TnI, sensitizes both the Tn complex and thin filaments to Ca2+, as well as slows both systems' Ca2+ dissociation rates  and (2) the effective concentration of TnI for the reconstituted thin filament was raised from 8 to 25 μM. Thus, our model predicts that unlike TnI S23/24D, TnI S150D increases the effective concentration of TnI that TnC “sees” when incorporated onto the thin filament. One possibility for this observation and a rationale for having to increase the effective concentration of TnI for TnC on the thin filament could be a weakened binding affinity of TnI S150D for actin. Consistent with this prediction, Salhi et al. has demonstrated TnI S150D has a weaker affinity for actin, compared to TnI S23/24D, which had a similar actin affinity as compared to wild type TnI  can be simulated with a simple two state system  both in the steady-state and kinetically. It is clear that Ca2+ triggers extensive dynamic changes in Tn that can be altered by disease associated mutations  why the Tn complex has such high apparent Ca2+ sensitivity and slow Ca2+ dissociation (compared to the isolated protein) that is drastically reduced and accelerated respectively, when the Tn complex is incorporated onto the thin filament. Figure 2+ sensitivity and dissociation rate of isolated TnC is modulated by the concentration of TnI. Furthermore, at saturating TnI concentrations, both the apparent Ca2+ sensitivity and dissociation rate are very similar to the intact Tn complex as well as the chimeras. Figure (2) Why a large proportion of Tn modifications seem to have no effect on the apparent Ca2+ binding properties of the isolated Tn complex, yet differences emerge when placed in the context of the thin filament. Figure 2+ binding properties of several Tn modifications can be modeled solely by changing the effective concentration of TnI on the thin filament. Furthermore, at a saturating TnI concentration, our model predicts for each of these Tn modifications that the apparent Ca2+ binding properties of the Tn complex are identical to that of the wild type Tn complex. Figure (3) There are several different molecular mechanisms within and outside of the Tn complex that influence the intrinsic and/or apparent Ca2+ binding properties of TnC. Our study focused on how altering the effective concentration of TnI can regulate the apparent Ca2+ binding properties of TnC. As pointed out in the manuscript and our model, we do not exclude that there are mutations/modifications in the Tn complex  that can alter the apparent Ca2+ binding properties of TnC though alternate mechanisms, such as altering the intrinsic rates of Ca2+, Mg2+, and/or TnI binding/dissociation from TnC. In fact, this is the point of a companion paper to be published in this thematic issue by the Biesiadecki lab  set by the compilation of proteins in the thin and thick filaments, as well as the effective concentration of TnI, work together to set the overall Ca2+ sensitivity of TnC. (4) At least four states of TnC are required to simulate the apparent Ca2+ binding properties of TnC in different experimental and diseased conditions. Figures We suggest the tethering of TnC to TnI and our model can help to explain: 2+ binding properties as well as evoking the concept of effective concentration. Considering nearly all of the myofilament protein-protein interactions that influence muscle contraction occur within confined and restricted spaces, effective concentration concepts may need to be invoked to simulate several other key reaction steps as well, especially myosin with actin , AG051913 (JD), HL117034 (ST), HL113084 (PJ), HL1114940 (BB), HL62426 (Pd).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


AML) comprises a range of disparate genetic subtypes, involving complex gene mutations and specific molecular alterations. Post‐translational modifications of specific proteins influence their translocation, stability, aggregation and even leading disease progression. Therapies that target to post‐translational modification of specific proteins in cancer cells represent a novel treatment strategy. Non‐homogenous subcellular distribution of PLSCR1 is involved in the primary AML cell differentiation. However, the nuclear translocation mechanism of PLSCR1 remains poorly understood. Here, we leveraged the observation that nuclear translocation of PLSCR1 could be induced during wogonoside treatment in some primary AML cells, despite their genetic heterogeneity that contributed to the depalmitoylation of PLSCR1 via acyl protein thioesterase 1 (APT‐1), an enzyme catalysing protein depalmitoylation. Besides, we found a similar phenomenon on another AML‐related protein, N‐RAS. Wogonoside inhibited the palmitoylation of small GTPase N‐RAS and enhanced its trafficking into Golgi complex, leading to the inactivation of N‐RAS/RAF1 pathway in some primary AML cells. Taken together, our findings provide new insight into the mechanism of wogonoside‐induced nuclear translocation of PLSCR1 and illuminate the influence of N‐RAS depalmitoylation on its Golgi trafficking and RAF1 signalling inactivation in AML.Acute myeloid leukaemia ( Acute myeloid leukaemia (AML) is a clinically devastating disease characterized by the arrest of leukaemic blast at the early stages of differentiation and the retention of self‐renewal stage during leukaemic myeloblast development via a labile thioester bond Reagents targeting the post‐translational modification of critical pathogenic proteins in AML malignancy, including the farnesyltransferase inhibitor (FTI), ubiquitin–proteasome inhibitor and histone deacetylase inhibitor (HDACI), have been the focus of intense interest for drug development. Thereinto, palmitoylation is a post‐translational lipid modification involving the attachment of a 16‐carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins Previous investigation demonstrated that phospholipid scramblase 1 (PLSCR1) expression was up‐regulated by wogonoside in 14 of 23 (~61%) of patients with AML [PBMC derived from four relapsed (RR) and 19 newly diagnosed (ND) patients were analysed for PLSCR1 expression using Western blots], suggesting that PLSCR1 acts as an important effector in wogonoside‐treated primary AML cells Ras gene and high expression of oncogenic RAS proteins commonly exist in approximately 30% of human cancers, including myeloid malignancies Ras as a driver of pathogenesis in several human tumours According to the hypothesis that wogonoside may control the palmitoylation status of PLSCR1, thus facilitating its attachment to the specific plasma membrane compartments or nuclear transportation, we speculated that the effect of wogonoside on palmitoylation modification might be non‐specific. The small GTPase RAS is usually used as a paradigm to show how reversible acylation allows proteins to be trapped and released from specific membrane compartments. The mutational activation of Scutellaria baicalensis Georgi (huang qin), both in vivo and in vitro, and highlighted the nuclear translocation of PLSCR1 in wogonoside‐induced primary AML cell differentiation Our previous studies have demonstrated the antileukaemic properties of wogonoside, a flavonoid extracted from Wogonoside  was dissolved in dimethylsulphoxide (DMSO) as a stock solution at 0.5 M and stored at −20°C. Wogonoside stock solution was freshly diluted with medium to the final concentration (150 μM) before each experiment. The final DMSO concentration did not exceed 0.1%. Cells treated with the highest concentration of DMSO were used as control in the corresponding experiments.N‐Ethylmaleimide (NEM), hydroxylamine (HAM) solution, 4, 6‐diamidino‐2‐phenylindole dihydrochloride (DAPI), MTT  and cycloheximide (CHX) were purchased from Sigma‐Aldrich . Biotin‐BMCC, Streptavidin‐HRP and Western blot Stripping Buffer were purchased from Thermo Scientific .All‐trans retinoic acid (ATRA) was dissolved in DMSO as a stock solution at 0.01 M and used as an inducer of PLSCR1. A nuclear/cytosol fractionation kit  was used according to the manufacturer's directions. ® 488 goat anti‐rabbit IgG (H+L) antibody, Alexa Fluor® 488 donkey anti‐goat IgG (H+L) antibody and Alexa Fluor® 555 donkey antimouse IgG (H+L) antibody were purchased from Life Technologies .PLSCR1 (Human) IP‐WB antibody pair and APT1 antibody were purchased from Abnova . Primary antibody against β‐actin, laminA, N‐RAS and APT1 siRNA were obtained from Santa Cruz Biotechnology . Fluorescein isothiocyanate (FITC) anti‐human CD14 was obtained from Miltenyi Biotec Inc . Phycoerythrin (PE) anti‐human CD11b antibodies were obtained from eBioscience . Antibody against RAF1 and p‐RAF1 were products from Abclonal Biotechnology . Anti‐GM130 antibody was obtained from BD Biosciences . IRDyeTM 800‐conjugated secondary antibodies were purchased from Rockland . Alexa Fluoret al. 2 at 37°C.Human AML cell line U937 and HL‐60 were from Cell Bank of Shanghai Institute of Biochemistry & Cell Biology. Primary leukaemic cells from newly diagnosed AML patients without prior therapy  were collected using lymphocyte–monocyte separation medium . The protocol of collection of cells from patients complied with guidelines in the Declaration of Helsinki and was approved by The First Affiliated Hospital of Nanjing Medical University's institutional review board and the appropriate ethics committees. A signed informed consent was obtained from each patient. Primary leukaemia cell isolation was performed as described previously in Hussong via IP ABE assay is the most ideal method for measuring the dynamic turnover of palmitate on proteins Cells were plated in six‐well plates with fresh RPMI 1640 medium. siRNA transfection was performed with Lipofectamine 2000 reagent according to the manufacturer's instructions 5 cells.Fluorescence intensity of CD11b and CD14 was analysed with a FACSCalibur flow cytometer  Primary AML cells were collected at various time‐points after wogonoside (150 μM) treatment. A nuclear–cytosol fractionation kit used the nuclear and cytosolic protein extraction preparations according to the modified method as described Preparation of whole‐cell lysates was performed as described previously ® 555 Donkey antimouse IgG (H+L) antibody (1:500) and Alexa Fluor® 488 Goat anti‐Rabbit IgG (H+L) antibody (1:500) for 1 hr at 37°C. Then, the coverslips were washed and counterstained with DAPI working solution (100 μg/ml) for 20 min. at room temperature. The coverslips were inverted onto slides and immersed in a mounting medium. The images were captured with a confocal microscope at 1000× magnification .Cells were collected onto the coverslips and fixed in ice‐cold methanol for 10 min. Then coverslips were permeabilized in 0.2% (v/v) Triton X‐100 for 20 min. and blocked with BSA buffer (PBS containing 3% BSA) for 1 hr at room temperature. Next, cells were co‐incubated with GM130 (1:100) and primary anti‐N‐RAS antibody (1:50) and then 4°C overnight. The coverslips were washed three times and followed by co‐incubation with Alexa Fluoranova) followed by the Bonferroni post hoc test. Comparisons between two groups were analysed using two‐tailed Student's t‐tests. A P value < 0.05 was considered statistically significant.All data were expressed as mean ± SD from at least three independent experiments performed in a parallel manner. Statistical analysis of multiple‐group comparisons was performed by one‐way analysis of variance  treatment not only increased the expression of PLSCR1 but also promoted its translocation to nucleus in primary AML cells , we were intrigued whether the nuclear translocation of PLSCR1 was due to the up‐regulation of PLSCR1 expression. To further ascertain this reasoning, #1 primary AML cells were pre‐treated with ATRA for 48 hrs, and then, the cells were pre‐treated with 15 μg/mL cycloheximide (CHX), the protein synthesis inhibitor, followed by 150 μM wogonoside for 24 hrs and 48 hrs. ATRA induced the up‐regulation of PLSCR expression in a time‐dependent manner without distribution priority, suggesting that ATRA can elevate PLSCR1 expression but cannot promote its nuclear translocation in primary AML cells  is a 29‐kDa cytosolic protein that exhibits lysophospholipase activity towards palmitoylated glycerol‐3‐phosphocholine and can remove palmitate from proteins on the cytosolic surface of membranes via APT1 in AML cell lines and primary AML cells. We speculated that the effect of wogonoside on post‐translational modification might be non‐specific. It is well known that N‐RAS is a palmitoylated and hyperactivated kinase in AML From the results above, we found that wogonoside promoted the depalmitoylation of PLSCR1 The activation of small GTPase RAS and its kinase cascade RAF1 contributes to a number of cellular processes including cell proliferation, survival, differentiation and apoptosis Although the contribution of PLSCR1 nuclear translocation to wogonoside‐induced differentiation of AML cells has been reported The palmitoylation state has been known to determine the subcellular localization of PLSCR1, and depalmitoylated PLSCR1 can be imported into the nucleus for binding genomic DNA Protein palmitoylation and depalmitoylation have been proven to be a reversible process regulated by two types of enzymes, acyltransferases (PATs) and protein thioesterases (APT1 and PPT1) Multiple lines of research on the regulation of RAS focused on the post‐translational modifications, including the constitutive and irreversible remodelling of its carboxy‐terminal CAAX motif by farnesylation, proteolysis and methylation, reversible palmitoylation and conditional modifications PLSCR1 and N‐RAS signalling play important roles in the progression of AML malignancy. Our previous investigation focused on the effect of wogonoside on the PLSCR1 in 23 AML clinical samples. Samples were divided into three types according to their different responses to wogonoside in PLSCR1 expression Targeting the post‐translational modifications of these proteins is a new strategy for the design and development of anti‐AML drugs. This study demonstrated that wogonoside promotes both PLSCR1 nuclear translocation and Golgi trafficking, which are responsible for its antileukaemia activity, by inducing PLSCR1 depalmitoylation and N‐RAS depalmitoylation, respectively. These findings suggest the potential role of wogonoside as a novel agent in AML treatment.H.L. designed and performed research, analysed data and wrote the manuscript; X.Y. and X.L. performed research. P.H. and L.S. analysed data; Y.Z. performed the animal experiments; J.X. and H.H. collected data and performed statistical analysis; Y.Z. provided the blood samples; Z.L. analysed the compound; Q.G. and J.X. conceptualized the project and directed experiment design and data analysis.The authors declare no conflict of interest.Figure S1  Immunofluorescence of 150 μM wogonoside‐treated primary AML cells (#5 and #6) for 48 hrs was performed. Cells were collected and were co‐stained with anti‐GM130 (primary)/Alexa Fluor® 555 Donkey anti‐Mouse (secondary) antibody (red fluorescence) combinations anti‐N‐RAS (primary)/Alexa Fluor® 488 Goat anti‐Rabbit (secondary) antibody (green fluorescence), as well as DAPI (blue fluorescence). They were detected by confocal microscopy  with FV10‐ASW2.1 acquisition software (Olympus) at room temperature . Images are representative of three independent experiments.Click here for additional data file.Figure S2 Growth inhibition effects of wogonoside on AML cells and human normal peripheral blood mononuclear cells (PBMC). Primary AML cells  and PBMC were incubated in 96‐well plates with 5 × 104/well in 100 μL culture medium, then were treated with 100 μL various concentration of wogonoside for 96 hrs, respectively. Cell viability was determined using MTT assay. Data were shown as means ± SD (n = 3).Click here for additional data file. Click here for additional data file.