Fig 1: Pyridostigmine reduces STIM1‐dependent store‐operated Ca entry (SOCE) in HF myocytes. (A) Representative images of Fluo‐4 fluorescence illustrating SOCE response in cardiac myocytes from CTL, TAC and TAC + PYR groups. Myocyte sarcoplasmic reticulum (SR) Ca was depleted by 1 µM thapsigargin, a SR Ca ATPase inhibitor, and SOCE was induced by rapid increase in extracellular Ca from 0 to 2 mM. (B) Representative time‐courses of spatially averaged fluorescence before and following application of Ca were recorded in myocytes from CTL, TAC, and TAC + PYR groups. (C) Summary data on the amplitude of local Ca increase in CTL, TAC and TAC + PYR myocytes, 1‐way ANOVA + Tukey HSD. (D) Summary data for the fraction of cells exhibiting SOCE response in CTL, TAC and TAC + PYR groups, respectively. (E) Boxplot illustrating maximal changes in myocyte fluorescence in response to application of external Ca, 1‐way ANOVA + Tukey HSD. For SOCE experiments, n = 11‐34 cells per group, n = 3‐10 mice per group, minimum of three experiments
Fig 2: Inhibition of SOCE increases Homer1a expression.PC12 cells were trasfected with STIM1 specific siRNA (Si-STIM1) or control siRNA (Si-Control) 72 h before MPP+ insult, or pretreated with ML-9 (50 μM) or MRS (15 μM) 30 min before MPP+ insult, and the expression levels of Homer1a mRNA (A) and protein (B) were detected by Real-time RT-PCR or Western blot, respectively. The data were represented as means ± SD from five experiments. * p < 0.05 vs. MPP+ alone. # p < 0.05 vs. control siRNA.
Fig 3: STIM1 colocalizes with BK and TRPM4.(A) Total internal reflection fluorescence (TIRF)-mode superresolution localization maps of freshly isolated vascular smooth muscle cells (VSMCs) from control mice immunolabeled for BK (red) and STIM1 (green). Colocalized BK and STIM1 clusters were identified by object-based analysis (OBA) and mapped (cyan). Scale bar: 3 µm. Panels to the right show enlarged areas of the original superresolution maps indicated by the white boxes. Arrows show examples of colocalizing clusters. Scale bar: 500 nm. (B) TIRF-mode superresolution localization maps of freshly isolated VSMCs from control mice immunolabeled for TRPM4 (cyan) and STIM1 (magenta). Colocalized TRPM4 and STIM1 clusters were identified by OBA and mapped (yellow). Scale bar: 2 µm. Panels to the right show enlarged areas of the original superresolution maps indicated by the white boxes. Arrows show examples of colocalizing clusters. Scale bar: 500 nm. (C) Colocalization frequency of BK and STIM1 clusters in imaged cells compared to colocalization frequency of BK and STIM1 clusters in randomized maps generated from respective cells. n = 11 cells from four mice (*p<0.05, paired t-test). (D) Colocalization frequency of TRPM4 and STIM1 clusters in imaged cells compared to colocalization frequency of TRPM4 and STIM1 clusters in randomized maps generated from respective imaged cells (n = 11 cells from four mice; *p<0.05, paired t-test). Figure 5—source data 1.Individual data points and analysis summaries for datasets shown in Figure 5.
Fig 4: Stim1 knockout decreases colocalization of TRPM4 and IP3R protein clusters.(A) Epifluorescence-mode superresolution localization maps of freshly isolated vascular smooth muscle cells (VSMCs) from control and Stim1-smKO mice immunolabeled for TRPM4 (cyan) and IP3R (magenta). Colocalized TRPM4 and IP3R clusters were identified by object-based analysis (OBA) and mapped (yellow). Scale bar: 3 µm. Panels to the right show enlarged areas of the original superresolution maps indicated by white boxes. Scale bar: 500 nm. (B) Summary data showing the density (clusters per unit area), frequency distribution of sizes, and mean size of TRPM4 channel protein clusters. (C) Summary data showing the density, frequency distribution of sizes, and mean size of IP3R clusters. (D) Summary data showing the density, frequency distribution of sizes, and mean size of colocalizing TRPM4 and IP3R clusters, identified using OBA. For density data, n = 15 cells from three mice for both control and Stim1-smKO mice. For frequency distribution and mean cluster size data: control, n = 64,292 TRPM4 channel clusters, n = 51,728 IP3R clusters, and n = 5164 colocalizing clusters; Stim1-smKO mice, n = 56,771 TRPM4 channel clusters, n = 45,717 IP3R, and n = 3981 colocalizing clusters (*p<0.05, unpaired t-test). ns, not significant. Figure 4—source data 1.Individual data points and analysis summaries for datasets shown in Figure 4.
Fig 5: UNC93B1 deficiency reduces STIM1–ORAI1 interactions.A, representative confocal images of WT and UKO cells stained with DAPI (blue) and the Duolink proximity ligation assay using mouse aSTIM1 and rabbit aORAI1 antibodies (red) before and 10 min after 1 µM Tg addition. B, numbers of immunoreactive dots per cell revealed by the proximity ligation assay. Data are mean ± SD of 22 images from six samples per condition. NS; **p > 0.01, two-way ANOVA (interaction: SS = 96.70, DF = 1, MS = 96.70, F(1,84) = 2.758, p = 0.1005). C, representative TIRF images of WT and UKO cells expressing Orai1-YFP (green) and mCherry-STIM1 (red) treated with 1 µM Tg for 10 min. D, Manders 1 and 2 colocalization index between Orai1-YFP and mCherry-STIM1 before and after Tg addition. Data are mean ± SD of 36 images from three independent experiments. NS; ***p > 0.005; ****p > 0.001, two-way ANOVA (M1 interaction: SS = 0.04521, DF = 1, MS = 0.04521, F(1,79) = 7.136, p = 0.0092; M2 interaction: SS = 0.06389, DF = 1, MS = 0.06389, F(1,79) = 5.191, p = 0.0254). E, time course of Tg-induced changes in mCherry-STIM1 TIRF fluorescence in WT and UKO cells. DAPI, 4',6-diamidino-2-phenylindole; NS, nonsignificant; STIM1, stromal interaction molecule 1; Tg, thapsigargin; TIRF, total internal reflection fluorescence; UKO, UNC93B1-deficient cell.
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