Figure 2.

LE/lysosome repositioning during ER stress is dependent on the degradation of Blos1 mRNA by RIDD. (A) We transfected MC3T3-E1 cells expressing LAMP1-GFP with siRNAs targeting UPR effectors or control siRNAs (Neg), then treated with Tg (2 µM, 12 h). Where indicated, we also added the IRE1 inhibitor 4μ8c (60 µM) 5 min before Tg. We then imaged and scored cells for juxtanuclear LE/lysosome clustering. (B) We collected RNA samples in parallel with A and measured Blos1 mRNA by qPCR. (C–G) We treated MC3T3-E1 cells stably expressing Rfp, Blos1^s-HA, or Blos1^s-Flag with Tg (2 µM), Tm (6 µg/ml), or serum-free media (S; 18 h). We then analyzed LE/lysosome clustering by LAMP1 immunostaining and Blos1 mRNA levels by qPCR. Panel E shows representative images, scale bar = 10 µm. (H and I) We incubated MC3T3-E1 cells stably expressing Rfp or Arl8b–Rfp with Tg (2 µM, 18 h), then analyzed as in C and D. *P < 0.05, stressed knockdown or overexpression cells versus controls, determined by ANOVA followed by Tukey’s honestly significant difference (A and B), or paired t tests corrected for multiple comparisons (C, F, and H), n = 3 except in A and B where the number of experiments is indicated by the symbols. UT, untreated; OE, overexpressed.