Fig. 2: Chloroquine, hydroxychloroquine, and azithromycin induce ER stress.

A, B Human osteosarcoma U2OS cells were treated with chloroquine (CQ), hydroxychloroquine (HCQ), and azithromycin (AZT; all at 10, 20, 40 μM) for 16 h, then fixed and imaged. Tunicamycin (TM, 3 μM) and thapsigargin (TG, 3 μM) were used as positive controls for ER stress induction. PeIF2α was assessed by means of an immunofluorescence staining using a phosphoneoepitope-specific antibody (A) and the cytoplasmic intensity is depicted (B) (AZT, CQ, and HCQ, 40 µM). C, D Human osteosarcoma U2OS cells stably expressing GFP under the promoter of DDIT3 (CHOP::GFP) were treated with the indicated agents (TM (3 μM), TG (3 μM), CQ, HCQ, or AZT (all at 0.1, 0.3, 1, 3, 10, 30 μM)) for 24 h. After fixation, CHOP::GFP fluorescence was assessed microscopically as shown in C, and the average nuclear intensity was quantified (D). E, F U2OS cells stably expressing GFP-ATF6 were treated with the indicated agents for 24 h. After the cells were fixed, GFP-ATF6 nuclear translocation was assessed as shown in E (AZT, CQ, and HCQ, 30 µM), and the nuclear-to-cytoplasmic ratio of GFP-ATF6 intensity was quantified (F). G, H U2OS cells stably expressing XBP1ΔDBD-venus (for monitoring venus expression upon alternative splicing of XBP1 mRNA) were treated as above for 24 h. After fixation, XBP1s expression was assessed via fluorescent microscopy as shown (G) (AZT, CQ, and HCQ, 30 µM), and the average intensity was measured (H). Data are means ± SD of four replicates (*P < 0.05, **P < 0.01, ***P < 0.001 vs. vehicle control (Ctrl), Student’s t-test). Scale bars equal 10 μm.