Figure 6. Abrogation of µ_s disposal through ERAD leads to BiP being permanently eclipsed, ER homeostatic failure, and apoptosis.

(A,D,E) HeLa-µ_s cells, harboring APEX-KDEL (D,E) or not (A) were induced with (µ_s) or without (ctrl) 0.5 nM Mif for 3 days in the presence or absence of 30 µM Kif. (A) Percentages of Annexin V positive cells were assessed by cytometric analysis. Mean and s.e.m. are shown in a bar graph, n = 2–4. (B,C) HeLa-µ_s cells were induced to express µ_s for various times as indicated (B) or for 3 days in the absence or presence of 30 µM Kif. (C) Levels of µ_s, BiP, and α-tubulin as well as activation of the IRE1α and PERK branches of the UPR were assessed as in (Bakunts et al., 2017). (B) Levels of BiP and µ_s were assessed by quantitative immunoblotting as described (Bakunts et al., 2017), and depicted in bar graphs as in Figure 2D,E, such that the µ_s levels in the absence of Kif were scaled to BiP levels at a ratio of 2:3. Levels in the presence of Kif are expressed as a fold change compared to levels in the absence of Kif; mean and s.e.m. are shown; n = 2. (D) In cells harboring APEX-KDEL the extent of ER expansion was assessed as in Figure 2A. Boxed areas are shown by 3-fold magnification; scale bars represent 1 µm. The percentage of the dark area within the cytoplasm corresponding to ER was determined and depicted in bar graphs (E), mean and s.e.m. are shown, n = 10. Statistical significance of differences in Annexin V staining (A), or the extent of ER occupying cytosolic area in the electron micrographs (E) were tested by ANOVA (*p≤0.05; ***p≤0.001).