Fig. 3: Chloroquine, hydroxychloroquine, and azithromycin-induced autophagy depends on TFEB/TFE3 and eIF2α.

A, B Human osteosarcoma U2OS wild type (WT) or TFEB/TFE3 double KO (TF DKO) cells both stably expressing GFP-LC3 were treated with the indicated compounds (torin 1 (TOR; 300 nM), chloroquine (CQ), hydroxychloroquine (HCQ), and azithromycin (AZT; all at 0.1, 0.3, 1, 3, 10, 30 μM)) for 6 h. After fixation, GFP-LC3 dots were analyzed as a proxy for autophagy. Representative images are depicted in A (AZT, CQ, and HCQ, 30 µM) and normalized data are shown as bar chart in B. Data are means ± SD of four replicates (**P < 0.01, ***P < 0.001 vs. WT; Student’s t-test). C, D U2OS WT or PeIF2α S51A knockin cells both expressing RFP-LC3 were treated as indicated with TOR (300 nM), bafilomycin A1 (BafA1, 100 nM), CQ, HCQ, and AZT (all at 10, 20, 40 μM) for 6 h. After fixation, RFP-LC3 dots were analyzed by fluorescent microscopy. Representative images are shown in C (AZT, CQ, and HCQ, 40 µM) and normalized data are quantitated as a bar plot in D. Data are means ± SD of four replicates (**P < 0.01, ***P < 0.001 vs. WT; Student’s t-test). Scale bars equal 10 μm.