Fig. 1

TMCO1 knockdown leads to the overactivation of IRE1α. a–b qRT-PCR analysis of Xbp1s/Xbp1u (a), and Edem1 (b) mRNA levels in WT or TMCO1 KD cells. Data are shown as mean ± SEM from three independent experiments. n.s., no significance, *P < 0.05, ** P < 0.01. c Western blotting analysis of the UPR marker proteins, including p-PERK, PERK, p-eIF2α, eIF2α, ATF4, p-IRE1α, IRE1α, XBP1s and ATF6-N (N-terminal cleavage product of ATF6), in untreated or TG-treated (1 μM, 4 h) WT and TMCO1 KD cells. GAPDH was used as a loading control. Relative quantification of each protein was shown in the right panel. Bar graphs represent the mean ± SEM from three independent experiments. *P < 0.05, **P < 0.01, n.s., no significance. d Western blotting analysis of proteins in WT and TMCO1 KD cells treated with/without 20 μM KIRA6 for 24 h. GAPDH was used as a loading control. e Relative quantification of p-IRE1α protein levels in D. Data are shown as the mean protein intensity normalized to GAPDH ± SEM from 3 independent experiments. n.s., no significance, **P < 0.01. f qRT-PCR analysis of Xbp1 mRNA splicing in WT and TMCO1 KD cells treated with/without 20 μM KIRA6 for 24 h. Bar graphs represent the mean ± SEM from three independent assays. **P < 0.01, n.s., no significance. g FACS analysis of mCherry-positive cells. WT or TMCO1 KD cells were transfected with the UPRE-mCherry. SSC-A: side-scatter area, which can indicate the granularity of the cell. Bar graph in the right panel represents the mean ± SEM for mCherry-positive cells from three independent assays. ***P < 0.001. h Western blotting analysis of p-IRE1α and IRE1α levels in WT and TMCO1 KD cells transfected with control mCherry vector or TMCO1-IRES-mCherry. Tubulin was used as a loading control. Relative quantification of protein levels is shown in the right panel. Data are shown as the mean protein intensity normalized to tubulin ± SEM from 3 independent experiments. *P < 0.05, **P < 0.01