Kinetochores link chromosomes to spindle microtubules and are essential for accurate chromosome segregation during cell division. Kinetochores assemble at the centromeric region of chromosomes as a multiprotein complex. However, the molecular mechanisms of kinetochore assembly have not yet been fully elucidated. In this study, we identified pleckstrin homology domain and leucine-rich repeat protein phosphatase 1 (PHLPP1) as a regulatory phosphatase that facilitates proper kinetochore assembly. We found that PHLPP1 interacted with the essential outer-kinetochore protein SGT1 and stabilized its protein levels. Loss of PHLPP1 from cells led to SGT1 degradation and thereby caused defective kinetochore assembly. We also found that the ring finger protein 41 (RNF41) as an E3 ligase ubiquitinated and degraded SGT1 in a phosphorylation-dependent manner. PHLPP1 dephosphorylated SGT1 at four conserved residues  and thereby prevented SGT1 from associating with RNF41, in turn, countering SGT1 degradation. Importantly, depletion of RNF41 or expression of a non-phosphorylatable SGT1 mutant rescued the kinetochore defects caused by the loss of PHLPP1. Taken together, our results suggest that PHLPP1 plays an important role in the assembly of kinetochores by counteracting RNF41-mediated SGT1 degradation. During cell division, accurate chromosome segregation is dependent on correct attachment of chromatids to spindle apparatus . KinetocHere, we identified PHLPP1 (pleckstrin homology domain leucine-rich repeat protein phosphatase 1) as an essential protein required for proper assembly of kinetochores in cells. PHLPP1 is a highly conserved serine/threonine phosphatase, which belongs to PPM family of phosphatases . PHLPP1 7–In our earlier study , we foun13–In this study, we found that SGT1 protein levels are tightly controlled in cells by RNF41-mediated ubiquitination. RNF41  is a ring type E3 ubiquitin ligase that has been implicated in controlling various cellular pathways, such as the EGF receptor, cytokine receptor, and T cell receptor signaling through ubiquitination of different substrates in these pathways –19. Howe–A). Furthermore, we found that SGT1 interacts with PHLPP1 but not with phosphatase and tensin homolog, another phosphatase in the PI3K/AKT pathway thus validating the specificity of the PHLPP1–SGT1 interaction .In an attempt to identify new cellular functions of PHLPP1, we analyzed the list of its interacting proteins derived from biochemical purification followed by proteomic analysis in our earlier study . We founFig. 1C) leads to delayed progression of cells in mitosis (D). Although control cells complete mitosis within 60 min after rounding up, cells expressing PHLPP1 shRNA spent several hours in mitosis before they aberrantly exit without dividing or undergoing cell death. Furthermore, live cell analysis using U2OS cells stably expressing H2B mCherry revealed that PHLPP1-depleted cells are predominantly delayed in the prometaphase stage of mitosis (E). Delayed progression of cells in mitosis upon PHLPP1 depletion (A) is accompanied by multiple severe mitotic defects such as misaligned chromosomes (B), multipolar spindles (C), and abnormal centrosomes (D). Next, we tested the effect of PHLPP1 depletion on direct assembly of kinetochores by staining with specific markers. By using confocal immunofluorescence imaging, we found that outer kinetochore proteins such as HEC1 (A) and CENP-E (B) failed to localize to kinetochores in PHLPP1-depleted cells. In contrast, localization of core inner kinetochore protein CENP-A is unaffected by PHLPP1 loss (C). As depletion of PHLPP1 caused severe reduction in recruitment of outer kinetochore proteins, we next tested whether kinetochore–microtubule attachment is affected in these cells. Indeed, co-staining of kinetochores and microtubules with CENP-A and α-tubulin, respectively, revealed that PHLPP1 depletion led to defective attachment of microtubules with kinetochores (D). Thus, together these data suggest that PHLPP1 is necessary for proper assembly of kinetochores.Because SGT1 is critical for proper kinetochore assembly during the mitotic cycle, we next tested whether loss of PHLPP1 phenocopies SGT1 loss from cells. Time-lapse imaging revealed that silencing of PHLPP1 in HeLa cells C leads t mitosis D. Althou mitosis E. Delayeepletion A is accoomosomes B, multipspindles C, and abtrosomes D. Next,  as HEC1 A and CENd CENP-E B failed PP1 loss C. As deptochores D. Thus, A). This result prompted us to test whether SGT1 protein stability is controlled by PHLPP1. In fact, in a cycloheximide chase experiment, shRNA-mediated knockdown of PHLPP1 reduced the protein half-life of SGT1 to a greater extent suggesting that PHLPP1 is required for maintaining the stability of SGT1 (B). The reduction in SGT1 levels upon PHLPP1 depletion was rescued in the presence of the proteasome inhibitor MG132. Because polyubiquitination is known to regulate protein stability through the proteasome, we further tested whether PHLPP1 maintains SGT1 stability by controlling ubiquitination. Knockdown of PHLPP1 resulted in accumulation of polyubiquitinated SGT1 (C), suggesting that PHLPP1 is required for preventing SGT1 ubiquitination.To further understand how PHLPP1 participates in kinetochore assembly by interacting with SGT1, we tested SGT1 localization on kinetochores. Immunofluorescence studies suggested that upon PHLPP1 depletion SGT1 is lost from the kinetochores A. This r of SGT1 B. The reted SGT1 C, suggessupplemental Table S1). Among these, we found RNF41 as a potential E3 ligase interacting with SGT1. We validated the interaction of SGT1 with wild-type (WT) RNF41 as well as a catalytically inactive mutant of RNF41 (C34A) in cells through co-immunoprecipitation experiments (A). Next, to test whether RNF41 can ubiquitinate SGT1, we performed ubiquitination assay in cells by co-expressing SFB SGT1 along with Myc-tagged RNF41 wild type and also the catalytically inactive mutant of RNF41. We observed that SGT1 was polyubiquitinated by wild-type RNF41 but not the catalytically inactive mutant (B). Polyubiquitination of SGT1 is through degradative Lys-48-linked ubiquitin chains as RNF41 failed to ubiquitinate SGT1 in the presence of ubiquitin K48R mutant (C). Because we identified RNF41 as an E3 ligase for SGT1 and PHLPP1 impedes SGT1 polyubiquitination, we hypothesized that PHLPP1 might prevent SGT1 from interacting with its E3 ligase RNF41. To test this hypothesis and to understand the interplay between RNF41 and PHLPP1 in regulating SGT1, co-immunoprecipitation was performed either after depleting PHLPP1 from cells or by exogenous expression of PHLPP1. Interaction of SGT1 with RNF41 is dramatically enhanced in the absence of PHLPP1 (D), and conversely exogenous expression of PHLPP1 leads to loss of SGT1 interaction with its E3 ligase (E). Thus, PHLPP1 protects SGT1 from polyubiquitination and degradation by interfering with SGT1 interaction with its E3 ligase RNF41.Although we found that PHLPP1 controls SGT1 polyubiquitination, the enzymatic machinery that mediates SGT1 ubiquitination in the cells is unknown. Thus, next we sought to identify E3 ubiquitin ligase for SGT1. In an attempt to identify the E3 ligases for SGT1, we established a 293T derivative cell line expressing a triple-epitope eriments A. Next, e mutant B. PolyubR mutant C. Becausf PHLPP1 D, and co3 ligase E. Thus, in vitro phosphatase assay, we found that wild-type PHLPP1, but not the PHLPP1 phosphatase-inactive mutant (D901N), readily dephosphorylated SGT1 (A). Furthermore, by using an in vitro pIMAGO-based detection of phosphorylation on recombinant proteins, we found that active PHLPP1, but not PTEN, dephosphorylates SGT1 thus confirming the specificity of PHLPP1-mediated dephosphorylation (B). By using mass spectrometric analysis, we found that human SGT1 was phosphorylated at five different residues  in the cell . We mutated each of these residues to alanine and tested the ability of PHLPP1 to dephosphorylate SGT1. We did not find any significant differences in the phosphate release by PHLPP1 using SGT1 single residue mutants. We assumed that PHLPP1 dephosphorylates multiple residues on SGT1. To test this possibility, we made several SGT1 mutants with mutations at these five residues in different combination. Indeed, we found that mutation of four residues Ser-17, Ser-249, Ser-289, Thr-233 (SGT1 4A) significantly lowered the release of phosphate from SGT1 by PHLPP1 (C). Furthermore, we confirmed the dephosphorylation of these sites in cells by expressing a SGT1 4A mutant in the presence of PHLPP1 shRNA. Although depletion of PHLPP1 caused enhanced phosphorylation of wild-type SGT1, no changes in the phosphorylation status of 4A mutant were observed (D).To understand the mechanistic details of how PHLPP1 prevents SGT1 from interaction with RNF41, we next tested whether SGT1 acts as a substrate of PHLPP1. By using an ted SGT1 A. Furtherylation B. By usiy PHLPP1 C. Furtheobserved D.E). Furthermore, RNF41 could not ubiquitinate a phospho-dead SGT1 4A mutant, but a significant accumulation of polyubiquitinated protein was observed with phosphomimetic SGT1 4E mutant  (F). Consistent with this, in a cycloheximide chase experiment, we observed that SGT1 4A mutant levels were stabilized compared with wild type, but on the other hand, the SGT1 4E mutant was highly unstable (G). Together, these results fully support our hypothesis that RNF41 interacts with SGT1 in a phosphorylated state, and PHLPP1 opposes RNF41 activity toward SGT1 by dephosphorylation.Next, we tested whether phosphorylation of these residues is required for SGT1-RNF41 interaction, which is opposed by PHLPP1. Our co-immunoprecipitation experiments suggested that wild type, but not a phospho-dead 4A mutant, of SGT1 interacts with RNF41 E. Furtheic acid) F. Consisunstable G. TogethA). During the later stages of mitosis, post-kinetochore assembly, interaction between PHLPP1 and SGT1 is lost, which then leads to RNF41 association and degradation of SGT1. The regulation of SGT1 by PHLPP1 and RNF41 appears to occur in the soluble pool of the cell but not on chromatin, because we did not observe any localization of RNF41 and PHLPP1 to chromatin (B). To further test whether the existence of interplay between PHLPP1 and RNF41 is functionally relevant for kinetochore assembly, we performed double depletion experiments. We hypothesized that SGT1 loss and kinetochore defects caused by PHLPP1 depletion would be rescued by simultaneous depletion of RNF41. In support of this hypothesis, co-depletion of RNF41 and PHLPP1 rescued SGT1 loss as well as recruitment of other proteins such as HEC1 (C) and CEPN-E (D) at kinetochores. In addition, overexpression of the non-phosphorylatable SGT1 4A mutant, but not phosphomimetic SGT1 4E mutant, also rescued the outer kinetochore assembly defects (A and B), thus fully supporting our data that PHLPP1-mediated dephosphorylation of SGT1 is critical for proper kinetochore assembly. Interestingly, expression of the SGT1 4A mutant also rescued spindle defects (C) as well as centrosome defects (D) caused by PHLPP1 depletion, possibly suggesting that SGT1 is a major player in PHLPP1 controlled mitotic processes. Indeed, these data are in agreement with earlier studies where SGT1 was shown to be required for centrosome organization  persist at centromeres during the entire cell cycle, the outer kinetochore proteins such as the KMN network that is composed of KNL1, the Mis12 complex, and the NDC80 complex are recruited specifically during Gs CENP-T , which is CENP-T . Aurora s CENP-T . AlthougIn this study, we provided multiple lines of evidence supporting an essential role of a phosphatase PHLPP1 in kinetochore assembly by regulating SGT1 protein levels. Previous studies showed that SGT1 plays an essential role in kinetochore assembly, but mechanisms to control its functions at kinetochores are not well defined. Previously, CK2 (casein kinase 2) was shown to negatively regulate SGT1 by phosphorylating it at serine 361 (in yeast), thus inhibiting SGT1 dimerization and affecting the kinetochore assembly . In contIn contrast, all the tumor suppressor activities of PHLPP1 have so far been attributed to its role in inhibiting various proliferation and survival signaling pathways. Our study provides an additional mechanism for PHLPP1 tumor suppressor function where loss of PHLPP1 resulted in multiple mitotic defects that subsequently lead to genomic instability. Further studies are required to uncover other possible substrates of PHLPP1 in mitosis, which may help in substantiating the role of PHLPP1 in controlling genomic stability.Full-length PHLPP1, SGT1, RNF41, and PHLPP1 devoid of the phosphatase domain were cloned into SFB triple-tagged (S-protein/FLAG/streptavidin-binding protein) and Myc destination vectors using the Gateway cloning system (Invitrogen). PHLPP1 was also cloned into an HA destination vector. The point mutations for PHLPP, SGT1, and RNF41 were generated by PCR-based site-directed mutagenesis. SGT1 plasmid was a kind gift from Dr. Anna Filipek. Human RNF41 cDNA was procured from the DNASU repository.Anti-PHLPP1 (A300-660A from Bethyl Laboratories and 07-1341 from Millipore), anti-SGT1 (A302-944A from Bethyl Laboratories and Ab30931 from Abcam), anti-RNF41 (A300-048A from Bethyl Laboratories), anti-CENP-A (Ab13939 from Abcam), anti-CENP-E (Ab5093 from Abcam), anti-HEC1 (Ab3613 from Abcam) anti-Myc, clone 9E10 (SC-40 from Santa Cruz Biotechnology), anti-histone H3 (05928 from Millipore), anti-γ-tubulin (T5326 from Sigma), anti-α-tubulin (T6074 from Sigma), anti-FLAG (F3165 from Sigma), anti-actin (A5441 from Sigma), anti-HA (A190-108A from Bethyl Laboratories), and anti-ubiquitin (05-944 from Millipore) antibodies were used in this study.m Tris-HCl, pH 8.0, 100 mm NaCl, 1 mm EDTA, 0.5% Nonidet P-40) containing 50 mm β-glycerophosphate, 10 mm NaF, 1 μg/ml of each pepstatin A, and aprotinin on ice for 30 min. After removal of cell debris by centrifugation, the cell lysates were incubated with streptavidin-Sepharose beads (Amersham Biosciences) for 1 h at 4 °C. The bound proteins were washed three times with NETN and then eluted with 2 mg/ml biotin (Sigma) for 60 min at 4 °C. The eluates were incubated with S-protein-agarose beads (Novagen) for 1 h at 4 °C and then washed three times with NETN. The proteins bound to S-protein-agarose beads were eluted by boiling in SDS-loading dye and then resolved by SDS-PAGE. The identities of eluted proteins were revealed by mass spectrometry analysis performed by the Taplin Biological Mass Spectrometry Facility at Harvard University.SGT1-associated proteins were isolated by using tandem affinity purification as described before . BrieflyHEK293T and HeLa cells were maintained in RPMI 1640 medium supplemented with 10% FBS and 1% penicillin and streptomycin, and the cells were transfected with various plasmids using polyethyleneimine transfection reagent. For immunoprecipitation assays, cells were lysed with NETN buffer. The whole-cell lysates obtained by centrifugation  were incubated with 2 μg of specified antibody bound to either protein A or protein G-Sepharose beads (Amersham Biosciences) for 1 h at 4 °C. The immunocomplexes were then washed with NETN buffer four times and applied to SDS-PAGE. Immunoblotting was performed according to the standard protocols.m) for 6 h. The whole-cell extracts prepared by NETN lysis under denaturing conditions were subjected to immunoprecipitation of the substrate protein. The levels of ubiquitinated protein were then detected by immunoblotting with ubiquitin antibody.HEK293T and HeLa cells were transfected with various combinations of plasmids, and 24 h after transfection, cells were treated with MG132 (10 μThe vector containing PHLPP1 shRNA-1 (5′-CCAGACTTACTACATTTGCTT-3′), PHLPP1 shRNA-2 (5′-CGAGGTCTTTCCCGAAGTTAT-3′), and RNF41 shRNA  was added. Cells were harvested at different time points, and protein levels were determined by immunoblotting after loading an equal concentration of protein per lane.in vitro kinase reaction was performed by incubating SGT1 with 1 mm ATP, HEK293T cell lysate, and reaction buffer  at 37 °C for 90 min. Following the incubation, beads were washed twice with NETN buffer. The phosphorylated SGT1 was incubated with bacterially purified MBP PHLPP1 WT and PHLPP1 D901N mutant in reaction buffer  at 30 °C for 60 min. The released phosphate was detected using malachite green assay kit (Cayman) by measuring the absorbance at 620 nm.GST SGT1 was bacterially purified, and an in vitro by using 293T cell lysate. The phosphorylated SGT1 was incubated with bacterially purified phosphatases  for 30 min. Later the samples were boiled, run on SDS-PAGE, and followed by transfer onto PVDF membrane. After incubating the membrane with blocking buffer (1 h), pIMAGO-Biotin reagent  was added and further incubated for 1 h. After three washes (5 min each) with wash buffer, the membrane was incubated with avidin-HRP for 1 h. After washing three times with 1× TBST, phosphorylation signals were detected by ECL reagent. The phosphorylation status of SFB-tagged SGT1 wild type and SGT1 4A mutant in cells was also detected by immunoblotting with pIMAGO reagent.GST-SGT1 was bacterially purified and phosphorylated m HEPES, pH 7.4, 50 mm NaCl, 3 mm MgCl2, and 300 mm sucrose at room temperature for 5 min, and cells were incubated with 1% BSA for blocking at room temperature for 60 min. After this step, cells were washed with PBS and incubated with primary antibodies for 2 h at room temperature followed by washing twice with 1× PBS. Cells were then incubated with FITC or rhodamine-conjugated secondary antibodies at room temperature for 60 min followed by washing twice with 1× PBS. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). After a final wash with PBS, coverslips were mounted with glycerin-containing para-phenylenediamine, and imaging was done using a confocal microscope .HeLa cells were grown on coverslips and fixed with 3% paraformaldehyde solution in PBS at room temperature for 15 min. Permeabilization of the cells was carried out with 0.5% Triton X-100 buffer containing 20 mHeLa cells were transfected with control and PHLPP1 shRNA, and 72 h after the transfection, cells were arrested using thymidine for 16 h. Later the cells were released from thymidine using fresh medium, and then image acquisition was performed with a Nikon A1R microscope equipped with a ×20 lens and enclosed in a chamber to maintain temperature for live cells. During imaging, cells were maintained in RPMI 1640 medium at 37 °C, and images were acquired at 1-min intervals with Nikon software. U2OS cells stably expressing H2B-mCherry were used in time-lapse imaging for mitotic stage-specific experiments.p values were determined by Student's t test using GraphPad software.Data from at least three sets of independent experiments were plotted in the graphs by including standard deviation. The S. M. and N. R. G. designed the experiments, analyzed the data, and wrote the manuscript. N. R. G. performed all the experiments.


Cox2, Bmp2, and Wnt4, and blocks embryo implantation. In Ishikawa cells, a human endometrial epithelial cell line, LEFTY2 downregulated the expression of calcium release-activated calcium channel protein 1, encoded by ORAI1, and inhibited store-operated Ca2+ entry (SOCE). Furthermore, LEFTY2 and the Orai1 blockers 2-APB, MRS-1845, as well as YM-58483, inhibited, whereas the Ca2+ ionophore, ionomycin, strongly upregulated COX2, BMP2 and WNT4 expression in decidualizing HESCs. These findings suggest that LEFTY2 closes the implantation window, at least in part, by downregulating Orai1, which in turn limits SOCE and antagonizes expression of Ca2+-sensitive receptivity genes.Early embryo development and endometrial differentiation are initially independent processes, and synchronization, imposed by a limited window of implantation, is critical for reproductive success. A putative negative regulator of endometrial receptivity is LEFTY2, a member of the transforming growth factor (TGF)-β family. LEFTY2 is highly expressed in decidualizing human endometrial stromal cells (HESCs) during the late luteal phase of the menstrual cycle, coinciding with the closure of the window of implantation. Here, we show that flushing of the uterine lumen in mice with recombinant LEFTY2 inhibits the expression of key receptivity genes, including •Endometrial receptivity is negatively regulated by LEFTY2.Cox2, Bmp2and Wnt4, and blocks embryo implantation.•LEFTY2 inhibits the expression of key murine receptivity genes, including •LEFTY2 downregulates the expression of Orai1 and inhibits SOCE.COX2, BMP2, and WNT4 expression in endometrial cells.•LEFTY2 and the Orai1 blockers 2-APB, MRS-1845, and YM-58483 inhibit •Targeting LEFTY2 and Orai1 may represent a novel approach for treating unexplained infertility.The online version of this article (10.1007/s00109-017-1610-9) contains supplementary material, which is available to authorized users. Lefty2 in the mouse uterus leads to implantation failure i) oscillations [2+]i is tightly regulated by several mechanisms, including Ca2+ release from intracellular stores and subsequent activation of store-operated Ca2+ entry (SOCE) iy (SOCE) . SOCE islum (ER) . Followilum (ER) . These rchannels , 27. Proonceptus . Recentlonceptus . Both Oronceptus . The rol2+]i levels in endometrial cells remain incompletely understood but presumably involve Ca2+ release from intracellular stores with subsequent activation of SOCE i(Δ[Ca2+]i) . Taken together, the data show that LEFTY2 blunts intracellular Ca2+release and SOCE.In order to explore whether LEFTY2 impacts on Ca]i) Fig. . Both peTY2 Fig. . Basal CLEFTY2, a member of the TGF-β superfamily, is implicated in embryo development and stem cell differentiation through its antagonistic action on the TGF-β/Smad signaling pathway –43. LEFT2+]i signaling, LEFTY2 decreases expression of Ca2+-responsive implantation genes such as COX2, WNT4 and BMP2. Other key implantation genes, including LIF, IHH, HOXA10 and HBEGF transcripts i to expression of receptivity genes requires further elucidation. The present observations uncover a novel function of Orai1 and store-operated Ca2+ entry. SOCE is known to trigger Ca2+ oscillations i i . Cells waddition . For qua of Ca2+ . ExperimFor histological assessment, uterine horns were formalin-fixed and embedded in paraffin, cut in 5-μm sections, and stained with H&E as previously described .t test or Kruskal-Wallis test was used when appropriate. Statistical significance was assumed when P < 0.05.Data were analyzed with the statistical package GraphPad Prism . Student’s ESM 1(PDF 217 kb)


In our previously study, up-regulated expression of transient receptor potential canonical channel (TRPC5) was proven to increase [Ca2+]i level, and resulted in chemoresistance whereas not apoptosis in human colorectal cancer (CRC) cells. The ATP-dependent homeostatic maintenance of resting [Ca2+]i should be important in this process. Increased glycolysis was found to be an important adenosine triphosphate (ATP) source in cancer. This study aimed to explore the potential mechanism of aerobic glycolysis in transient receptor potential channel TRPC5 induced chemoresistance.Elevated intracellular Ca2+ ([Ca2+]i) and the half maximal inhibitory concentration of 5-Fu (5-Fu IC50) were measured. Western blot was analyzed to determine cleaved Caspase-3 protein level. Flow cytometry was performed to detect the apoptosis rates. Immunohistochemistry staining was performed to determine TRPC5 and GLUT1 expression level in human CRC tissues.In this study, we examined glucose transporter 1 (GLUT1) expression, glucose consumption and celluar ATP production to determine glycolytic activity. Real-time PCR and western blot were analyzed to determine TRPC5 expression at the mRNA and protein levels in human CRC cells , and fluorouracil (5-Fu) resistant CRC cells . 3-bromopyruvate (3-BP) and 2-Deoxy-D-glucose (2DG) were used to inhibit glycolysis. Glycolytic activity, intracellular Ca2+]i levels which decreased after treated with TRPC5-specific shRNA. Furthemore, inhibition of glycolysis resulted in decreased ATP production, elevation of [Ca2+]i level and cleaved caspase-3, increased apoptotic cells rate, and a remarkable reversal of 5-Fu resistance in HCT-8/5-Fu cells, while showed no effect in HCT-8 cells. BAPTA-AM, a [Ca2+]i chelator, could reduce the elevation of cleaved caspase-3 and increased apoptotic cells rate due to glycolysis inhibition. Advanced CRC patients with high expression of TRPC5/GLUT1 displayed poorer chemotherapy outcome, and notably, the significant association between high TRPC5 expression and chemoresistance is GLUT1 expression level dependent.Overproduced of TRPC5 and increased glycolysis were found in HCT-8/5-Fu and LoVo/5-Fu than in HCT-8 and LoVo cells. Compared to HCT-8 cells, the HCT-8/5-Fu cells showed higher [Ca2+]i homeostasis.We demonstrated the essential role of glycolysis in TRPC5 induced chemoresistance in human CRC cells via maintaining [Ca Transient receptor potential canonical channel 5 (TRPC5) is a Ca2+-permeable channel that could modulate [Ca2+]i level. In our previously study [2+]i that could be increased by TRPC5, acts as “double-edged sword” in cellular process. At different levels, it not only participates in cell proliferation, differentiation and gene transcription, but also induces cell apoptosis [2+]i homeostasis might be important in TRPC5 induced chemoresistance.Colorectal cancer (CRC) is one of the most common malignant tumors and showed a high cancer-related death in China . Chemothpoptosis . Hence, 2+]i efflux is an energy dependent activity [2+]i efflux and in maintaining a low resting [Ca2+]i i , 11. HerThe wild human CRC cell line HCT-8 KG028) and LoVo (SCSP-514) were purchased from Keygen Biotech Co. Ltd.  and the Cell Resource Center of Shanghai Institutes for Biological Sciences, Type Culture Collection of the Chinese Academy of Sciences  respectively. Fluorouracil (5-Fu)-resistant HCT-8 cells (HCT-8/5-Fu) (KG333) was purchased from Keygen Biotech Co. Ltd. 5-Fu-resistant LoVo cells (LoVo/5-Fu) were derived by treating LoVo cells with stepwise increasing concentrations of 5-Fu  over 6 months. The wild human CRC cells and 5-Fu-resistant CRC cells were cultured as we reported previously . The bands were quantified using ImageJ software . β-actin was used as the internal control for normalization.TRIzol  was used to extract total RNA from cells. Real-time PCR and the comparision of the mRNA levels were performed according to the reported study i level. The fluorescence signals of GECO1.2 reflected the [Ca2+]i levels. The detailed procedure was in accordance with the previously reported study i level [2+ permeability [2+]i in HCT-8/5-Fu cells. In present study, according with the up-regulated expression of TRPC5, the level of [Ca2+]i in HCT-8/5-Fu cells was higher than in HCT-8 cells. Further inhibition of TRPC5 by shRNA resulted in decreased TRPC5 protein expression (Fig. 2+]i level (Fig. The roles of trp channels in cancer include changes in [Ca]i level . TRPC5 ieability . In our ion Fig.  along wivel Fig. .Fig. 2a 2+]i overload, which was demonstrated to induce apoptosis i overload related apoptosis. Since reprogramed energy metabolism to glycolysis was demonstrated to be the major mechanism of generating ATP [2+ efflux in cancer [2+]i homeostasis in chemoresistance induction by TRPC5. 3-BP and 2DG, inhibitors of glycolysis [2+]i level in HCT-8/5-Fu cells, while caused no obvious change in ATP production and [Ca2+]i level in HCT-8 cells (Fig. Several studies showed up-regulated expression of trp proteins  could lepoptosis , 19, 20.ting ATP , 7, 21 an cancer , we explycolysis , 22, werlls Fig. . In addills Fig. . FCM sholls Fig. . Furtherlls Fig. . MTT asslls Fig. . Similarlls Fig. .Fig. 3AdAs was shown in Table 2+ influx into cell, trp channels were demonstrated to be involved in many cellular biological behaviors in cancer i level was found to be positively associated with the TRPC5 level in chemoresistant CRC cells, which was up-regulated or decreased according to the TRPC5 expression. This indicated that TRPC5 regulates the cellular processes through alterring the Ca2+ influx. It has been demonstrated that [Ca2+]i is an important regulator of cell apoptosis at all stages [2+ efflux mechanism to maintain [Ca2+]i at a relatively high level not enough to trigger [Ca2+]i related apoptosis.In present study, the i efflux is an ATP-dependent process. In nonmalignant cells, oxidative phosphorylation is the main source of ATP under physiological condition, and inhibition of mitochondrial metabolism impaired [Ca2+]i homeostasis and leads to cell death [2+]i efflux and [Ca2+]i homeostasis in malignant cells i level, cleaved Caspase-3 and apoptotic cells rate, and reversed the resistance to 5-Fu in chemoresistant CRC cells, while did not cause significant change in wild human CRC cells. Since [Ca2+]i efflux is ATP-dependent, and elevated [Ca2+]i level has been proven to trigger apoptosis [2+]i level. In addition, the increased cleaved Caspase-3 and apoptotic cells rate induced by 2DG could be reduced by BAPTA-AM administration. This indicated the essential involvement of increased glycolysis in TRPC5 induced chemoresistance is [Ca2+]i homeostasis maintenance through supporting ATP. Further study on advanced CRC patients who received chemotherapy showed the impact of high TRPC5 expression on chemoresistance was high GLUT1 expression dependent.In this study, inhibition of glycolysis caused a remarkable ATP production decrease, increasement of i overload. Thus, rather than high TRPC5, high “TRPC5-glycolysis” was more closed to chemoresistance.In our previously study , 12, up-Aerobic glycolysis was proven to be crucial in tumorigenesis, tumor progression and metastasis , 34. Her


Coronal suture synostosis is a condition which can have deleterious physical and cognitive sequelae in humans if not corrected. A well-established animal model has previously demonstrated disruptions in intracranial pressure and developmental abnormalities in rabbits with congenital craniosynostosis compared to wild type rabbits.The current study aimed to measure the cerebral blood flow (CBF) in developing rabbits with craniosynostosis who underwent suturectomy compared to those with no intervention and compared to wild type rabbits.Rabbits with early onset coronal suture synostosis were assigned to have suturectomy at 10 days of age  or no intervention . A subset of each group was randomly selected for measurement at 10 days of age, 25 days of age, and 42 days of age. Wild type rabbits  were also randomly assigned to measurement at each time point as controls. Cerebral blood flow at the bilateral hemispheres, cortices, thalami, and superficial cortices was measured in each group using arterial spin-labeling MRI.At 25 days of age, CBF at the superficial cortex was significantly higher in EOCS rabbits (192.6 ± 10.1 mL/100 mg/min on the left and 195 ± 9.5 mL/100 mg/min on the right) compared to WT rabbits (99.2 ± 29.1 mL/100 mg/min on the left and 96.2 ± 21.4 mL/100 mg/min on the right), but there was no significant difference in CBF between EOCS-SU (97.6 ± 11.3 mL/100 mg/min on the left and 99 ± 7.4 mL/100 mg/min on the right) and WT rabbits. By 42 days of age the CBF in EOCS rabbits was not significantly different than that of WT rabbits.Suturectomy eliminated the abnormally increased CBF at the superficial cortex seen in EOCS rabbits at 25 days of age. This finding contributes to the evidence that suturectomy limits abnormalities of ICP and CBF associated with craniosynostosis. Craniosynostosis is a condition in which one or more of the calvarial sutures fuse prematurely. Though there are syndromic causes, most commonly, craniosynostosis is considered to be idiopathic, and its incidence is estimated at 1 out of 2500 live births. Involvement of the coronal sutures (CS) constitutes approximately 25% of cases  with conThe prevalence of intracranial hypertension in patients with craniosynostosis is unclear due to variation in method, duration of measurement, and ambiguity on what threshold value of intracranial pressure (ICP) is considered abnormal in each age group. Whereas reported prevalence ranges from 30–40% in syndromic craniosynostosis and 15–20% in nonsyndromic craniosynostosis, Tamburrini et al. (2005) argued for prolonged monitoring of ICP and attention to plateau waves and determined that 87.5% of syndromic patients with craniosynostosis had abnormal ICP . While dAlong with increased intracranial pressure (ICP) –13, otheConventional anatomical magnetic resonance imaging (MRI) allows for detection of structural changes in the brain in a non-invasive manner, but only after potentially irreversible damage has been done. Anatomical MRI does not allow for the study of CBF. The use of arterial spin labeling MRI (ASL-MRI) to serially and non-invasively study CBF represents a crucial adjunctive imaging modality.We have developed an inbred rabbit model of early-onset bilateral coronal craniosynostosis (EOCS) and have extensively described growth changes in the cranial vault and intracranial contents on these rabbits. Phenotypically, EOCS rabbits exhibit bony bridging of the coronal suture by 21 days gestation (gestation is 30 days in the rabbit), obliterated coronal sutures at birth, coronal ridging and brachycephalic cranial vaults by 10 days of age, and secondary changes in the cranial vault base, brain, and intracranial volume by 42 days of age , 21–24. The present study was undertaken to serially map CBF in our animal model and determine the impact of suturectomy on CBF in EOCS subjects. We used ASL-MRI to show the age-related changes in CBF, comparing wild-type subjects, and EOCS subjects with and without suturectomy over various time points. In light of the previous studies showing age-related increased ICP and CBF in rabbits with craniosynostosis and the prevention of early increased ICP via suturectomy , 24, 25,This study used a breeding colony of New Zealand white rabbits with naturally occurring coronal suture synostosis , 21–25. Synostotic rabbits were initially diagnosed at 10 days of age using previously described criteria , 21–24. Removal of the coronal suture (EOCS-SU group) occurred immediately after diagnosis of synostosis in a random subset of EOCS rabbits. The coronal suture was removed by drilling 2mm anterior to, and 2mm posterior to the suture, for the length of the affected suture . A 4mm xRabbits were randomly assigned into the following experimental groups: wild-type control rabbits (WT) at 10 days of age (n = 6); EOCS rabbits at 10 days of age (n = 6); EOCS-SU rabbits at 10 days of age (n = 5); WT rabbits at 25 days of age (n = 6); EOCS rabbits at 25 days of age (n = 6); EOCS-SU rabbits at 25 days of age (n = 5); WT rabbits at 42 days of age (n = 6); EOCS rabbits at 42 days of age (n = 6); and EOCS-SU rabbits at 42 days of age (n = 5).2O:O2 (1:2) gas mixture. The rabbits were placed in dorsal recumbency and an indwelling catheter was inserted into the femoral artery. Arterial blood gases were collected immediately before and after MRI. Arterial CO2 tension (PaCO2) was maintained between 20 and 46 mmHg for the duration of the study. This has been reported as the normal PaCO2 for rabbits [For magnetic resonance studies, at or near 10, 25, and 42 days of age, rabbits were transported to the Pittsburgh NMR Center for Biomedical Research at Carnegie Mellon University. Rabbits were preanesthetized with an intramuscular injection of ketamine (100mg/ml) and xylazine (20 mg/ml) in a 9:1 solution at a dose of 0.6 mg/kg. All rabbits underwent tracheotomy and were ventilated  via an endotracheal tube with 1–1.5% isoflurane in a N rabbits . During Atlas of the Rabbit Brain and Spinal Cord [1obs) was measured from a series of spin-echo images  [o flip angle, eight averages, TR/TE = 100/9.6 ms, 256x256 matrix and spin-labeling applied at ±6 mm) [MRI studies were performed on a 4.7-T, 40-cm bore Bruker AVANCE-AV system, equipped with a 12-cm-diameter-shielded gradient insert and a 72-mm volume RF coil for 10 and 25 days of age. At 42 days of age, half-PCOS RF coil was used. T2-weighted spin-echo images were used to verify the position of the perfusion slice, which was based on Figure 70 in the nal Cord , and wernal Cord . The labt ±6 mm) .C − ML) x MC−1 were generated from the perfusion data. MC and ML are the magnetization intensities from the control image and labeled image, respectively. T1obs maps were generated from the series of variable TR images by a three-parameter, non-linear fit. Regional CBF was then calculated according to Zhang et al [.(T1obsx2α)−1 x (MC − ML) x MC−1, where λ is the blood–brain partition coefficient of water, with a spatially constant value of 0.9 mL/g assumed [All image processing was performed with the Bruker ParaVision 5.0 image analysis software. Pixel by pixel maps of . Post-hoc group comparisons were made, and the Bonferroni correction was used for the multiple comparisons. All differences were considered significant if p < 0.05.p < .001), as shown in Representative CBF maps for WT, EOCS, and EOCS-SU rabbits from the 10-day, 25-day, and 42-day groups are shown in Our data demonstrates that in our rabbit model of early-onset craniosynostosis, age-related changes in CBF occurred in the superficial cortex compared to control animals and that this change was not seen in rabbits with early-onset craniosynostosis that underwent suturectomy. The increased CBF that is seen in the superficial cortex at 25 days coincides with the period of increased ICP previously reported by our group , which cSubjects that underwent suturectomy did not show a similar pattern of increased CBF at the superficial cortex at the 25-day time point. This study adds new evidence to the existing sequelae of craniosynostosis in the rabbit model, which includes decreased brain growth  and intrBased on the results of ASL-MRI, we found that cortical, hemispheric, and thalamic CBF were not different in rabbits with early-onset craniosynostosis of the coronal sutures when compared to age-matched wild-type rabbits and rabbits with early-onset craniosynostosis who underwent suturectomy. The increase in all groups seen between 10- and 25-days of age represents the normal pattern of CBF increase seen in the immature brain –39; TuorWhereas a rationale for increased CBF from 10 days of age to 25 days of age in normal rabbits has been described, the reason for the marked change in CBF and ICP from abnormally high on day 25 to normal on day 42 in rabbits with craniosynostosis is not well understood. With cerebral autoregulation, one may expect elevated ICP from craniosynostosis to result in either preserved CBF if the cerebral perfusion pressure (CPP) lies within the autoregulatory range or decreased CBF if the ICP is elevated to the extent that CPP is below the autoregulatory range. However, the CBF was actually greater in craniosynostosis rabbits than in normal rabbits at 25 days of age, which may represent an upward shift of the autoregulation curve. Shifts in the autoregulation curve have been described in humans and animals with orthostatic changes in sympathetic tone related to central venous pressure , 41. AddHuman data is sparse with regards to the influence of surgical correction on CBF. A Japanese group published their results on the use of I-123-IMP single photon emission computed tomography (SPECT) imaging studies in thirteen patients with craniosynostosis before and after surgical correction, with eight patients showing a focal area of hypoperfusion on the preoperative study . FollowiASL-MRI represents a promising non-invasive imaging modality that could provide insight into the anatomical etiology of the neurodevelopmental deficits seen in patients with craniosynostosis. Furthermore, its ability to provide both quantitative and qualitative data with high spatial resolution represents key advantages over other imaging modalities. However, the reliability of the results of this study is limited by high variability from small sample sizes and the inability to test repeatability of CBF measures from ASL-MRI due to instrument decommissioning. Also, a comparison of CBF as measured by ASL-MRI in rabbits with unilateral coronal suture synostosis could add validity to the conclusion that CBF is abnormal in craniosynostosis if CBF were elevated in the same side of the brain as the fused suture.While direct neurohistopathological analysis was beyond the scope of the present study, previous results from this rabbit model have shown that early suturectomy resulted in the preservation of white matter integrity as assessed by diffusion tensor imaging (DTI) , an incrOur well-established rabbit model of familial coronal suture synostosis , 25, 51 Based on the result of this experiment alone, the influence of suturectomy on mitigating downstream functional deficits is unclear. Further studies with the rabbit model will have to be coupled with behavioral testing as well as histopathology. Ultimately, in the clinical setting, the use of imaging such as ASL-MRI, along with pre- and postoperative neuropsychological testing may provide further insight into the influence of surgical intervention in the neurocognitive development of children with craniosynostosis.Coronal suture synostosis is associated with aberrations of ICP and CBF in the developing brain. Using a well-established animal model of craniosynostosis and arterial spin-labeling MRI, CBF has been studied in the developing brain of rabbits with coronal suture synostosis. Previously, authors have demonstrated a two-fold increase in CBF in the superficial cortex at 25 days of age in EOCS rabbits compared to WT rabbits followed by a return to normal CBF at 42 days of age. The current study demonstrated that after coronal suturectomy in EOCS rabbits, the CBF at 25 days of age was similar to WT rabbits. These findings set the stage for further animal and human studies to determine whether there is a critical period during which suturectomy could minimize functional impairment secondary to craniosynostosis.S1 TableCerebral blood flow in mL/100g/min for each animal at each region of interest. Data for each age group are in successive tabs. The final tab shows mean cerebral blood flow in mL/100g/min for each group.(XLSX)Click here for additional data file.


The active-site mTOR inhibitors (asTORi) represent a promising new approach to targeting mTOR in AKT/mTOR signaling. MLN0128 is an orally-administered, second-generation asTORi, currently in clinical development. We examined the anti-leukemic effects and the mechanisms of action of MLN0128 in AML cell lines and primary samples, with a particular focus on its effect in AML stem/progenitor cells. MLN0128 inhibited cell proliferation and induced apoptosis in AML by attenuating the activity of mTOR complex 1 and 2. Using time-of-flight mass cytometry, we demonstrated that MLN0128 selectively targeted and functionally inhibited AML stem/progenitor cells with high AKT/mTOR signaling activity. Using the reverse-phase protein array technique, we measured expression and phosphorylation changes in response to MLN0128 in 151 proteins from 24 primary AML samples and identified several pro-survival pathways that antagonize MLN0128-induced cellular stress. A combined blockade of AKT/mTOR signaling and these pro-survival pathways facilitated AML cell killing. Our findings provide a rationale for the clinical use of MLN0128 to target AML and AML stem/progenitor cells, and support the use of combinatorial multi-targeted approaches in AML therapy. The AKT/mTOR signaling pathway regulates cellular growth, survival, and proliferation , 2. Dysrwww.clinicalTrials.gov) [mTOR acts in two distinct complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). mTORC1 promotes protein translation and synthesis by phosphorylation of the substrates 4EBP1 and S6 kinase; mTORC2 controls cell survival and proliferation through downstream activation of AKT and AGC protein kinase . The claals.gov) –22.Limited studies have been done to investigate the effects of mTORC1/C2 inhibition in AML , 23, parSeveral high-throughput technologies, such as mass cytometry time of flight (CyTOF)  and reveThe anti-leukemic efficacy of MLN0128 was examined in four AML cell lines: FLT3-ITD-mutated MOLM13 and MV4-11 cells; NPM1 and N-Ras-mutated OCI-AML3 cells; and in PTEN-null U937 cells. In a dose-dependent fashion, MLN0128 caused growth inhibition at low nanomolar concentrations, and induced apoptosis at higher concentrations Figure . A similTo confirm the on-target molecular mechanisms of MLN0128, we assessed inhibition of AKT/mTOR signaling in three AML cell lines  and primary AML samples by Western blot analysis. MLN0128 attenuated the activity of both mTORC1 and mTORC2 at nanomolar concentrations. MLN0128 inhibited AKT phosphorylation at the mTORC2 site Ser473 and reduced phosphorylation of AKT substrates PRAS40 and GSK. It also blocked phosphorylation of mTORC1 substrates S6 (Ser240/244) and the rapamycin-resistant site on p-4EBP1 (Thr37/46) Figure . ApoptosMLN0128 induced apoptosis in AML CD34+ cells, prompting us to investigate its effects in AML stem/progenitor subpopulations defined by CyTOF, a novel technology that incorporates cytometry and mass spectrometry to measure multiple parameters without spectral overlap. Analysis of the CyTOF data by the analytic tools Spanning-Tree Progression of Density-Normalized Events (SPADE)  and visuSPADE analysis of 3 AML samples identified 15 unique subpopulations (subsets) with various cell frequencies and differential expression of surface and intracellular markers, as summarized in the table and heatmap Figure . These tMLN0128 induced apoptosis in AML progenitor CD34+ cells carrying FLT3-ITD, promoting us to further investigate its effects on the function of FLT3-mutated leukemic stem cells. We performed a colony-forming assay on both primary and secondary xenografted cells derived from two AML samples carrying the FLT3-ITD mutation Figure . At 30 nWe next used CyTOF assay to investigate the inhibitory mechanisms of MLN0128 in AML-derived leukemic cells from spleen and BM of secondary xenograft AML (sample #4). Combined analysis with SPADE, viSNE, and FlowJo identified 16 subpopulations (subsets), which were again grouped based on CD34 expression: group 1, CD34 medium to high (4 subsets); group 2, positive to medium (6 subsets); group 3, low or no expression CD34 (6 subsets)  in five sample subgroups , indicating heterogeneous responses to MLN0128 among primary AML.Our observation that residual AML cells survived after MLN0128 treatment prompted us to investigate alterations in the parallel signaling networks induced by MLN0128 and to identify potential antagonistic mechanisms upon mTOR inhibition. Using RPPA, a high-throughput functional proteomic technology, we measured expression and phosphorylation changes in response to MLN0128 in 151 proteins from 24 primary AML samples with high blast count . StatistRPPA analysis identified three druggable resistance targets: HDAC3, p-Src, and STAT5. mTOR blockade by MLN0128 increased the expression of HDAC3 and of p-Src (Tyr416) in 12 samples, and of STAT5  in 18 of 24 samples. Concurrent activation of all three targets was observed in 8 samples Figure . These tTo further evaluate the combinatorial approaches in AML under the conditions mimicking the hypoxic bone marrow microenvironment, we treated OCI-AML3 and U937 cells and three primary AML samples at the fixed concentration of each agent in co-culture with mesenchymal stromal cells (MSC), under hypoxia (1% oxygen) or normoxia (21% oxygen) Figure . The micWe previously reported that the first-generation asTORi PP242 disrupts mTORC1/C2 in AKT/mTOR signaling, targeting AML cells under conditions that mimic the bone marrow microenvironment . In thisAML is organized in a hierarchy with a small population of leukemia stem cells that are resistant to chemotherapy and propagate disease . Using CUsing CyTOF we next characterized AML-derived xenograft leukemic cells carrying FLT3-mutation. The distinct surface and intracellular phenotype identified on the leukemic cells isolated from spleen and BM supports the hypothesis that the xenograft microenvironment (murine BM vs. spleen) influences leukemogenic polarization and lineage differentiation . FurtherRPPA data confirmed the inhibitory effect of MLN0128 on mTORC1/C2 substrates; it also revealed that MLN0128 inhibition suppresses FAK, TG2, and β-catenin, all of which are highly expressed in AML and are associated with drug-resistance , 43. FAKPathway analysis using RPPA data indicated antagonistic responses upon mTORC1/C2 blockade in several intracellular networks, including anti-apoptotic signaling network, DNA repair, mitochondrial maintenance, and stem cell preservation. Five correlation clusters were identified. Each containing key molecules from these compartments suggesting that these responses are network-connected. Furthermore, five sample subgroups each containing the five correlation clusters were identified, with distinct alteration patterns, indicating the presence of sample-specific response signatures. Together, our data demonstrate the cellular network complexity and heterogeneity of responses to mTORC1/C2 inhibition. A similar study in other types of cancer cells treated with the dual PI3K/mTOR inhibitor NVP-BEZ235 demonstrated the expected inhibition of mTORC1 targets of p-4EBP1 and p-S6. However, the survival mechanisms triggered by BEZ235 differed from those of MLN0128 in AML , suggestOur RPPA findings highlight the need to combine mTOR kinase inhibitors with other targeted agents that suppress survival signaling in AML. We identified three druggable targets that were induced upon mTORC1/2 inhibition. We showed that the pan-HDAC inhibitor vorinostat and MLN0128 induced apoptosis in the majority of AML cell lines and in primary AML samples in an additive or synergistic fashion. Several putative mechanisms for the synergistic nature of this combination were recently described in pediatric B-ALL, such as downregulation of FOXO transcription factors and enhanced mitochondrial priming . WhetherIn summary, our findings indicate that blocking AKT/mTOR signaling with the mTORC1/C2 inhibitor MLN0128 causes growth inhibition and apoptosis in AML cells. High-throughput CyTOF technology revealed that MLN0128 selectively targeted AKT/mTOR signaling in AML stem/progenitor cells. Mapping multiple intracellular signaling pathways in primary AML in response to mTORC1/C2 inhibition identified the compensatory activation of actionable pro-survival pathways. Combinatorial multi-targeted approaches demonstrated various responses in AML samples, suggesting that personalized treatment approaches are warranted. Finally, techniques such as CyTOF or RPPA may assist in profiling the intra-cellular and inter-patient variations in responses to targeted agents, which likely stem from multiple genetic and epigenetic events within individual tumors, and serve as tools for the selection of individualized targeted therapies in AML and other malignancies.Information about materials, reagents, cell lines and patient samples used in this study is provided in the Supplementary Materials and Methods section.Cell viability was measured by Vi-Cell XR cell viability analyzer  or Cell Titer-Glo® Luminescent cell viability assay  as indicated in the text. Apoptosis of AML cell lines and progenitor cells  was analyzed by flow cytometry of annexin V  and propidium iodide (PI) or DAPI positivity . The extent of drug-specific apoptosis was assessed by the formula: % specific apoptosis = (test − control)/(100 − control) × 100. Where “test” is the percentage of annexin V/PI or DAPI positivity in treated cells, and “control” is the percentage of annexin V/PI or DAPI positivity in untreated cells (spontaneous apoptosis) . For theProtein expression in drug-treated cells was determined by Western blot as described previously . ProteinPeripheral blood samples were collected from 24 AML patients with high blast count . Mice were sacrificed when 80 % of engraftment was achieved. Engrafted cells were isolated from mouse BM and spleen, then sorted on human CD45+/DAPI-. Sorted cells were used for colony formation and CyTOF assay.Serial transplantation of primary AML in NSG mice  was performed as described previously . Briefly2. AML blast colonies were assessed by light microscopy between days 8 and 10.The colony-forming assay was performed as described previously . Sorted The CyTOF assay was performed as described in a previous report  and in tt-test and one – way ANOVA followed by Tukey's post-hoc test were used to calculate p values for experiments with single-paired or multiple-paired comparisons. Statistical analyses associated with particular methods are indicated in the text. A p value ≤ 0.05 was considered statistically significant. All cell lines experiments were performed in triplicate unless stated otherwise.Student's Targeting of mTORC1/C2 by the active-site mTOR kinase inhibitor MLN0128 leads to anti-leukemic effects in AML and AML stem/progenitor cells.


Intracellular Ca2+ signaling (ICS) waves, which occur in glia and retinal cells in response to injury to activate cell regulatory pathways, have been proposed as an early event in cochlear injury. Disruption of ICS activity is thought to underlie Connexin 26-associated hearing loss, the most common genetic form of deafness, and downstream sequelae of ICS wave activity, such as MAP kinase pathway activation, have been implicated in noise-induced hearing loss. However, ICS waves have only been observed in neonatal cochlear cultures and are thought to be quiescent after the onset of hearing. In this study, we employ an acute explant model of an adult, hearing cochlea that retains many in vivo physiologic features to investigate Ca2+ changes in response to sound. We find that both slow monotonic changes in intracellular Ca2+ concentration as well as discrete ICS waves occur with acoustic overstimulation. The ICS waves share many intrinsic features with their better-described neonatal counterparts, including ATP and gap-junction dependence, and propagation velocity and distance. This identification of ICS wave activity in the adult, hearing cochlea thus confirms and characterizes an important early detection mechanism for cochlear trauma and provides a target for interventions for noise-induced and Connexin 26-associated hearing loss.Ca Acoustic trauma is among the most common causes of permanent sensorineural hearing loss . Though 2+ signaling (ICS) waves in this process. In response to mechanical trauma, hair-cell ablation, or exogenous ATP exposure, ATP is released through connexin-based hemichannels on the apical surface of supporting cells, activating apical purinergic receptors, intracellular Ca2+ second messengers, and release of Ca2+ from endoplasmic reticular stores i), ratiometric imaging was performed using FURA-2-AM, a cell-permeant dye, in neonatal organotypic cultures [2+]i, and was measured in regions of interest using MetaFluor. Calibration of [Ca2+]i to FURA-2 fluorescence ratio was performed using a set of calibration standard  in water; however, because in situ calibration could not be performed in the cochlear preparations, we only report relative changes in [Ca2+]i within a single experiment, and not absolute [Ca2+]i levels.To evaluate intracellular Cacultures  and adul2+-ATPase (SERCA), were bath applied.ATP was applied to specimens either by bath application, or by using a pneumatic PicoPump to deliver a puff of ATP through a pulled glass micropipette with a 10-μM tip. For direct mechanical damage, a glass micropipette was pulled with a 2–3 μm tip, positioned with a micromanipulator at the reticular lamina above an outer hair cell (OHC), and the back of the pipette holder manually tapped to elicit mechanical trauma. Carbenoxolone (CBX), a general inhibitor of connexin-based channels, apyrase, an ATP-degradation enzyme, gentamicin, an aminoglycoside blocker of hair-cell mechanoelectrical transduction, and thapsigargin, an inhibitor of sarco/endoplasmic reticulum Ca2+] changes in the presence of different drugs was performed in a unique freshly dissected cochlear explant, in order to avoid irreversible effects of acoustic overstimulation. For all measurements of sound-induced [Ca2+] changes, baseline 340/387 ratio was measured in the measurement medium  for 2 minutes prior to sound exposure. Sound was then applied for 300 s and 340/387 ratio continuously measured at 2-second intervals. 340/387 ratio values at -60 s, 0 s, +80 s and +300 s relative to sound initiation were calculated as the average of 5 timepoints immediately preceding each mark. The change in 340/387 ratio was then calculated as the difference between the -60-s and 0-s values , or the difference between the +80s or +300s value and the 0-s value , and reported as a velocity of change (change in 340/387 ratio per minute) to account for the different measurement intervals. The 80s (early) and 300s (late) values were then corrected for the baseline drift.Each measurement of sound-induced i , OHCs sometimes exhibited an increase in [Ca2+]i (3/5 preparations) and other times showed a decrease in [Ca2+]i upon sound stimulation. The rise in [Ca2+]i peaked in the first 80 s after initiation of sound, with a subsequent slower phase of increase or, sometimes, reversal back towards baseline; this late Ca2+ behavior was highly variable from one preparation to another  or direct mechanical damage to an OHC with a 2-3-μm micropipette elicited ICS waves  and 78.0 (56.7–99.3) μm vs 68.9 (51.8–86.0) μm (p = 0.53), respectively (N = 7 for each)). Peak fluorescence ratio (340/387) change, was significantly higher in the neonatal cultures (0.31 (0.20–0.42) vs 0.55 (0.42–0.68), respectively (p = 0.016); ICS wave velocity was measured by identifying the peak [Cacochleae . Wave prin vitro experiments in the adult gerbil cochlea described above suggested that acoustic overstimulation induces early changes in [Ca2+]i as well stochastic triggering of ICS waves that are both dependent upon extracellular ATP and sensitive to blockade by CBX. Individuals with isolated disruption of ICS activity but intact junctional conductance owing to V84L Cx26 mutation have hearing loss. We hypothesized that Cx26 deficiency could thus predispose to noise-induced hearing loss owing to disruption of ICS activity. To investigate this, we evaluated noise-induced shifts in hearing thresholds in a TMX-inducible conditional KO model of Cx26-associated hearing loss, which avoids the developmental consequences of Cx26 dysfunction while permitting the evaluation of its functional effects i, as well as ICS waves very similar to those seen in neonatal cochlear cultures, can be elicited in the presence of physiologic levels of acoustic overstimulation. Acoustic overstimulation induces changes in [Ca2+]i in all cell types; these changes are dependent upon extracellular ATP, connexin hemichannel release of ATP, and mechanoelectrical transduction, and occur within tens of seconds of sound exposure. Acoustic overstimulation or direct mechanical trauma also produces ATP-dependent, CBX-sensitive sporadic ICS waves in pillar cells that are very similar to those seen and previously characterized extensively in supporting cells in neonatal cochlear cultures. These findings provide proof of principle that the phenomenon of trauma-induced, ATP- and connexin-dependent ICS waves persists in the hearing cochlea with physiologic stimuli.Cair cells , the rol2+ waves seen in neonatal cultures, adult cochlear ICS waves were markedly less frequent and showed significantly less change in absolute [Ca2+]i; in fact, these waves were only infrequently captured even in the presence of acoustic overstimulation. This may be due to a greatly increased threshold for ATP-induced [Ca2+]I increase- though we were able to elicit increases in [Ca2+]I with exogenous ATP, high concentrations of ATP were required, in the tens of micromolar. This is in contrast to the [Ca2+] increases in tens of nanomolar measured in endolymph after sound overexposure in vivo I changes was lower in the adult cochleae.Compared to Ca as 2 mM . A chang2+ second messengers including IP3 through gap junctions between supporting cells, and release of Ca2+ from intracellular endoplasmic reticular stores  in cochlear cells upon acoustic overstimulation is largely consistent with prior observations of [Ca2+]i changes in an isolated guinea pig temporal bone preparation  is consistently seen early during sound stimulation in IHCs and pillar cells, findings in OHCs, and at later timepoints, are more variable. This is in contrast to prior findings of consistent OHC Ca2+ accumulation upon sound overstimulation i on the order of minutes after initiation of sound were highly variable. This experimental variability may be an artifact of the explant system, or may represent true physiologic variability. Evaluation of these homeostatic Ca2+ transients in the adult cochlea in vivo would be necessary to further explore their effect on downstream cell regulatory pathways.The organ of Corti functions in a highly regulated ionic and micromechanical environment that cannot be perfectly replicated, even with this two-chamber explant system; therefore, this study remains limited. ICS waves were not consistently observed, and late changes in [Ca2+ signaling in the cochlea as an early response to acoustic overstimulation is reminiscent of ICS waves in the glia and retina, which have been implicated in a wide array of sometimes contradictory downstream cell homeostatic and regulatory functions, including neuronal guidance, synaptic maintenance, apoptosis, and cellular regeneration [Nevertheless, this demonstration of sound-induced homeostatic Caneration . The lan


While much is known about the regulation of SOCE by STIM1, the role of stromal interaction molecule 2 (STIM2) in SOCE remains incompletely understood. Here, using clustered regularly interspaced short palindromic repeats -CRISPR associated protein 9 (CRISPR-Cas9) genomic editing and molecular imaging, we investigated the function of STIM2 in NIH 3T3 fibroblast and αT3 cell SOCE. We found that deletion of Stim2 expression reduced SOCE by more than 90% in NIH 3T3 cells. STIM1 expression levels were unaffected in the Stim2 null cells. However, quantitative confocal fluorescence imaging demonstrated that in the absence of Stim2 expression, STIM1 did not translocate or form punctae in plasma membrane-associated ER membrane (PAM) junctions following ER Ca2+ store depletion. Fluorescence resonance energy transfer (FRET) imaging of intact, living cells revealed that the formation of STIM1 and Orai1 complexes in PAM nanodomains was significantly reduced in the Stim2 knockout cells. Our findings indicate that STIM2 plays an essential role in regulating SOCE in NIH 3T3 and αT3 cells and suggests that dynamic interplay between STIM1 and STIM2 induced by ER Ca2+ store discharge is necessary for STIM1 translocation, its interaction with Orai1, and activation of SOCE.Store-operated calcium entry (SOCE), a fundamentally important homeostatic and Ca Ca2+ signals are generated by the mobilization of calcium into the cytosol via influx through Ca2+-permeable ion channels in the plasma membrane (PM) and following release of Ca2+ from sites of intracellular sequestration.Dynamic changes in the concentration of free calcium ions within the cytosol (ER) leads to the dissociation of Ca2+ from the Ca2+-binding helix-loop-helix structural domain (EF-hand motif) of STIM1, which consequently induces a conformational change and oligomerization of STIM1. This is followed by a rapid translocation and accumulation of STIM1 in discrete multi-protein clusters or punctae in plasma membrane-associated ER (PAM) nanodomains, subcellular regions in which the PM and ER membranes are in close apposition and functionally interconnected ER ER ,23,24. Is [2+]ER . 2+ homeostasis and signaling, we used clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genomic editing and molecular imaging to determine the roles of STIM2 in Ca2+ homeostasis and SOCE in NIH 3T3 cells, a fibroblast cell line derived from mice, and αT3 cells, an immortalized gonadotroph cell line from mice. Our hypothesis was that STIM2 would be important for both Ca2+ homeostasis and SOCE. Our studies confirmed that STIM2 participates in the regulation of basal [Ca2+]c in unstimulated NIH 3T3 and αT3 cells, and is necessary for activation of SOCE following depletion of ER Ca2+ stores. We also found that STIM2 was required for STIM1 translocation and puncta formation, as well as essential for the interaction of STIM1 with Orai1 in NIH 3T3 cells. Taken together, our findings suggest STIM2 recruits STIM1 and stabilizes it in PAM nanodomains to facilitate interaction with and activation of Orai1 channels.In order to provide a greater understanding of how STIM2 participates in Ca2+ stores with cyclopiazonic acid (CPA), an inhibitor of sarcoendoplasmic reticulum Ca2+-ATPase (SERCA), followed by addition of extracellular Ca2+, resulted in an increase in [Ca2+]c, characteristic of SOCE found in many cell types (N-(1-[4-hydroxy-2-(trifluoromethyl)benzyl]-1H-pyrazol-3-yl)benzamide] (GSK-7975A), a reagent that inhibits SOCE downstream of STIM1 oligomerization and STIM1-Orai1 interaction [2+]c during the first 60 s of Ca2+ addback by 95 ± 3%  during the incubation of cells in Ca2+-containing solutions was reduced by 97 ± 2% , an endocannabinoid that reversibly inhibits SOCE by disrupting STIM1-Orai1 interactions [2+] and AUC60 were reduced by 70 ± 2% and 71 ± 2%, respectively , a cation that enters and blocks the ion-conducting pore of non-selective channels, but not Ca2+ selective channels, had no effect on SOCE. This suggests that SOCE is mediated by a channel that exhibits Ca2+ selectivity in the NIH 3T3 fibroblasts  by targeting a sequence in exon 2  compared to WT cells   C. 2+]ER using D1ER, a fluorescence resonance energy transfer (FRET)-based indicator of [Ca2+]ER [p > 0.05) between the resting level of [Ca2+]ER in STIM2 knockout  and control  cells -N,N,N′,N′-tetraacetic acid (EGTA) to prevent the influx of extracellular Ca2+ across the plasma membrane. The ER is the major Ca2+ store in the cell; therefore, the normalized maximum Fura-2 FI340/FI380 (∆peak) of the Ca2+ response following application of ionomycin is an indirect measure of the amount of Ca2+ sequestered within the lumen of the ER. Using this approach, we found no difference in ER Ca2+ store content in STIM2 null cells  compared to WT   cells D. As an  n = 61) D. Taken Xenopus oocytes [2+]ER [2+]c amplitude and overall SOCE response was significantly reduced (p < 0.05) in the NIH 3T3 STIM2 knockout cells (KO2-1)  compared to WT  (n = 22)  . The inh(n = 22) . Stim2    compared to WT    D. Simila > 0.05) E. To com0 wells) F,G. The 2 wells) H. These 2+]c in the Stim2 null αT3 cells was 10% lower  than in control cells expressing a scrambled gRNA sequence (Scr-1) , and there was no difference in basal [Ca2+]ER  compared to control cells   F–H. Mult2+ stores refilled    to characterize the distribution of STIM1 before and after store depletion. Confirming previous reports ,9,44, YFrefilled A. In conrefilled B. The pu n = 18) D,E. When, n = 9) C–E. This2+ stores under basal, unstimulated conditions and cells treated with CPA to deplete [Ca2+]ER and activate SOCE were examined. A STIM1-specific antibody was used to pull down STIM1 and immunoprecipitates were analyzed by Western blot for STIM2 and Orai1. STIM2 was detected in the STIM1 immunoprecipitates in both the control and CPA-treated cell lysates; under ER store-depletion conditions, the interaction between STIM1 and STIM2 was enhanced  to stabilize STIM at ER-PM junctions c was measured using Fura-2. Cells were grown on glass coverslips and incubated 15 min at room temperature with 1 μM Fura-2 acetoxymethylester , 0.01% (v/v) Pluronic® F-127 in standard extracellular solution (SES) (NIH 3T3 cells) or Krebs-Ringer-HEPES buffer (KRH2) (αT3 cells). SES contained (in mM): 140 NaCl, 4 KCl, 1 CaCl2, 2 MgCl2, 1 KH2PO4, 10 HEPES-NaOH (pH 7.4), and 10 d-glucose. KRH2 contained (in mM): 140 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 10 HEPES-NaOH (pH 7.4) and 2 d-glucose. Cells were placed into a temperature-controlled microperifusion chamber of an inverted fluorescence microscope . Cells were continuously superfused with SES or KRH2 (2 mL/min) at 37 °C. Fura-2 excitation wavelengths were 340 nm and 380 nm. The fluorescence emission intensities detected at 510 nm during excitation of Fura-2 at 340 nm (FI340) and 380 nm (FI380) were measured. MetaFluor software  was used for image acquisition and analysis. Background fluorescence was subtracted from each FI340 nm and FI380 nm image. FI340/FI380 ratios were normalized to the average resting ratio 60 s prior to application of the stimulus. [Ca® F-127). The loading solution was removed and replaced with SES. [Ca2+]c measurements were made using a FlexStation 3 plate reader controlled with SoftMax Pro software (Molecular Devices). Cells were excited at 335 nm and 375 nm, and the emitted light was detected at 505 nm using a 435-nm dichroic mirror. Test solutions were added to individual wells using the automated injection function of the FlexStation 3 at time points indicated in the figures. Data are expressed as the ratio of FI335/FI375 or relative FI335/FI375 .The SOCE inhibitor studies and NMG experiments were performed in a 96-well format in a FlexStation 3 multi-mode microplate reader . NIH 3T3 cells  and αT3 cells  were seeded in a black-walled 96-well plate. Twenty-four hours later, cells were loaded with Fura-2 for 30 min at room temperature . A suspension of NIH 3T3 cells (5 × 106 cells/mL) was electroporated at 1350 V with two 20 ms pulses. Parameters for the αT3 cell electroporation were 1500 volts, 1 pulse, 20 ms. Cells were seeded and cultured on glass coverslips for 48 h at 37 °C, placed into a heated microperifusion chamber mounted on the stage of an inverted microscope (Olympus IX81) equipped with a charge-coupled device camera . Cells were superfused with SES or KRH2 at 37 °C and visualized with a 40× oil immersion objective. D1ER was excited at 440 nm and emission intensities were measured at 485 nm (FRET donor) and 535 nm (FRET acceptor). MetaFluor software  was used for image acquisition and analysis. Data are expressed as the fold-change in ratio of FRET acceptor and donor emission intensities (FRET FI535/FI485) normalized to resting, unstimulated values.Cells were transfected with a plasmid construct encoding D1ER, a fluorescence resonance energy transfer (FRET)-based indicator of c was measured. As an alternative strategy to indirectly measure ER CaTo measure the interaction of STIM1 and Orai1, cells were co-transfected with YFP-STIM1 (FRET acceptor) and CFP-Orai1 (FRET donor) using electroporation as described above. Quantitative FRET imaging was performed as described above. The WT cells served as our biological control, showing differences in the magnitude of FRET between Orai1-CFP and STIM1-YFP in WT cells compared to KO2-1. Additionally, a vehicle control (DMSO) was used to show no change in FRET when perifused onto cells instead of CPA. Data are expressed as the fold-change in the background subtracted ratio of FRET acceptor and donor emission intensities (FRET FI535/FI485 nm) normalized to resting values. Resting FI535/FI485 values in KO2-1 and WT were not different.2+ extracellular solution to establish a baseline distribution, and then superfused with a Ca2+-free solution supplemented with 20 μM cyclopiazonic acid (CPA) to discharge and prevent refilling of intracellular Ca2+ stores. To restore ER Ca2+ levels, cells were bathed in the normal extracellular solution without CPA. Confocal images acquired immediately before the reconstitution of extracellular Ca2+ and CPA washout were background subtracted and processed using 2D deconvolution. Morphometric analysis was used to count the number and determine the area of individual puncta in each cell .Cells were transfected with YFP-STIM1 in WT and KO2-1 cells. To reconstitute STIM2 expression in KO2-1 cells, YFP-STIM1 was co-expressed with CFP-STIM2. Two days post-transfection, a time-lapse series of images were acquired in which cells were perifused with test solutions at 37 °C. Cells were initially bathed in normal, 1 mM CaN-(1-[4-hydroxy-2-(trifluoromethyl)benzyl]-1H-pyrazol-3-yl)benzamide] (GSK-7975A) was obtained from Glaxo Laboratories Ltd. . N-arachidonoyl glycine (NAGly) was purchased from Cayman Chemical . Cyclopiazonic acid (CPA) was obtained from Millapore Sigma. Orai1-E106A, CFP-Orai1 and YFP-STIM2 were obtained from Addgene.org. YFP-STIM1 was a generous gift from Tobias Meyer (Stanford University). D1ER was provided by the late Roger Y. Tsien . Fura-2 acetoxymethylester and ionomycin were purchased from Thermo Fisher Scientific and CalBiochem, respectively. ML-9 and SKF96365 hydrochloride were obtained from Sigma-Aldrich Co. . [2,6-difluoro-t-test assuming equal variance for intergroup comparisons , as detailed in the figure legends. Data traces are plotted as the mean ± SEM. Box and whisker plots show mean (open square), median (solid line), 25th and 75th percentiles (box), and minimum and maximum measurements (whiskers).Our data was analyzed and revealed nearly normal to normal distribution, equal variance, and were independent measurements; therefore, we used unpaired Student’s