Fig 2: Rescue of DYNC1LI2 expression reduces ER stress in ctns-/- cells. (A) The expression level of the established UPR target gene HSPA5, which is induced during conditions of ER stress, was analyzed in wild-type (WT), ctns-/-, and GFP-DYNC1LI2-expressing WT and ctns-/- fibroblasts by western blotting, using a mouse monoclonal anti-KDEL antibody. (B) Quantification of HSPA5 expression levels from 3 independent experiments. (C) HSPA5 expression in wild-type, ctns-/-, and GFP-DYNC1LI1-expressing WT and ctns-/- fibroblasts by western blot. (D) Quantification of HSPA5 expression levels from 3 independent experiments. Mean ± SEM, **, p < 0.01; NS, not significant. In Figure 4A and C, HSPA5 and ACTB were detected on the same membrane. (E) Representative immunoblot analysis of the indicated ER stress markers. DDIT3 and ACTB are probed in the same membrane. Total protein loading controls for all proteins are provided in Figure S7. (F-I) Quantification of the expression of EIF2A (F), its phosphorylated form (G), ATF4 (H) and DDIT3 (I). The data is presented as Mean ± SEM from three independent experiments. (J and K) Representative images and quantitative immunofluorescence analysis of the expression of ATF4 (J) and DDIT3 (K) in WT, ctns-/-, and GFP-DYNC1LI2-expressing ctns-/- cells. A total of 21 to 35 (ATF4) and 39 to 45 (DDIT3) independent images were quantified for each cell type in three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, not significant.

Fig 3: Proposed mechanisms of mutated hERG-induced cell apoptosis. Mutated L539fs/47-hERG protein retained in the ER lumen provokes ERS. Elevated expression levels of GRP78 activate the PERK-eIF2a-CHOP pathway, which is important in the mechanism of L539fs/47-hERG-induced ERS-mediated cell apoptosis. ER-specific caspase-12 is involved in the apoptotic mechanisms caused by the hERG mutation through cleaving and activating caspase-3. The pair of apoptosis factors Bax/Bcl-2 also regulate L539fs/47-hERG-induced cell apoptosis. hERG, human ether-à-go-go-related gene; WT, wild-type; 4PBA, 4-phenyl butyric acid; ER, endoplasmic reticulum; ERS, ER stress; ATF6, activating transcription factor 6; IRE1, inositol-requiring enzyme 1; p-PERK, phosphorylated protein kinase R-like endoplasmic reticulum kinase; eIF2a; eukaryotic translation-initiation factor-2a; CHOP, C/EBP homologous protein; Bcl2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.

Fig 4: Maytansine treatment promotes the phosphorylation of EIF2A in target cell lines.BT474 (a) and BJAB (b) cells in culture were either untreated (top) or treated with free maytansine (100 nM, bottom) for the times indicated. Cells were directly lysed into Laemmli buffer containing DTT, transferred to PVDF membranes, and probed by immunoblotting for EIF2A total protein (eIF2a) or Ser51 phosphorylated-EIF2a (P-eIF2A).

Fig 5: GSH prevented ERS responses propagated by SFN in FHL124 cells. Cells were maintained in either serum-free (control) EMEM or EMEM supplemented with 1-mM GSH. Following a period of 1 hour, half of the preparations were treated with SFN (final concentration 50 µM) and the other half did not receive SFN treatment. (A) Gene expression of two ERS gene markers (ERN1, encoding IRE1, and DDTI3, encoding CHOP) was measured after 18 hours of SFN treatment using TaqMan quantitative RT-PCR. Protein levels of two ERS protein markers (phosphor-EIF2? and XBP-1) were examined after 18 hours of SFN treatment using western blot. (B) Representative gel from the western blot. (C) Quantification of the western blot data adjusted for ß-actin loading controls. Quantitative data were pooled from four (A) or three (B, C) separate experiments and are shown as mean ± SEM. Asterisks indicate a significant difference between the treated group and untreated controls (P = 0.05, ANOVA with Tukey's post hoc test).