(A,D,E) HeLa-µs cells, harboring APEX-KDEL (D,E) or not (A) were induced with (µs) 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-µs cells were induced to express µs for various times as indicated (B) or for 3 days in the absence or presence of 30 µM Kif. (C) Levels of µs, 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 µs were assessed by quantitative immunoblotting as described (Bakunts et al., 2017), and depicted in bar graphs as in Figure 2D,E, such that the µs 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).