Fig. 3

Over-activation of IRE1α induced by TMCO1 knockdown is dependent on Ca²⁺ rather than the TMCO1 protein. a Representative images of Fura-2 340/380 ratios in WT, TMCO1 KD cells and TMCO1 KD cells transfected with TMCO1 or TMCO1-D140A-IRES-mCherry. Ratios were recorded for 1 μM TG-induced Ca²⁺ transients in these cells. The pseudo-color calibration scale for 340/380 ratios was shown on the right. mCherry images (first column) were captured before TG-induced Ca²⁺ imaging to identify TMCO1 expressing cells. Representative images were shown for cells 30 s before (second column), and 45 s, 75 s, 120 s, and 240 s after application of 1 μM TG as indicated. Scale bar, 20 μm. b Left panel, 1 μM TG-triggered Ca²⁺ transients in WT transfected with IRES-mCherry (purple trace line, n = 124), TMCO1 KD transfected with IRES-mCherry (red trace line, n = 76), TMCO1 KD cells transfected with TMCO1-IRES-mCherry (green trace line, n = 62), TMCO1 KD cells transfected with TMCO1-D140A-IRES-mCherry (blue trace line, n = 173). Each trace line in B is an average of Ca²⁺ responses from all mCherry-positive cells in each group. Right panel, statistical analysis of the average peak area of 1 μM TG-triggered Ca²⁺ mobilization curves. Bar graphs represent the mean ± SEM from three independent assays. ***P < 0.001. c Western blotting analysis of p-IRE1α, IRE1α and XBP1s levels in WT, TMCO1 KD cells, TMCO1 KD cells transfected with TMCO1 or TMCO1-D140A-IRES-mCherry. GAPDH was used as a loading control. Relative quantification of each protein levels was shown in the right panel. Bar graphs represent the mean ± SEM from three independent experiments. **P < 0.01, ***P < 0.001, n.s., no significance. d qRT-PCR analysis of Xbp1 mRNA splicing in WT, TMCO1 KD HeLa cells, TMCO1 KD HeLa cells transfected with TMCO1 or TMCO1-D140A-IRES-mCherry. Bar graphs represent the mean ± SEM from three independent assays. **P < 0.01, ***P < 0.001, n.s., no significance