Fig. 2: Lysosomal inhibition increases ATF4 accumulation but also limits ATF4 binding to the promoters of UPR^mt genes.

a Western blot analysis showing time-dependent changes of proteins in MEFs pretreated with DMSO control or ConA (200 nM) for 1 h, and then co-treated with Dox (30 μg/mL) for 0–8 h. All ConA-treated conditions were thus treated with ConA for a total time of 9 h. b Western blot analysis of MEFs treated with ConA (200 nM) or Torin1 (250 nM) for 0–24 h. c Western blot analysis of MEFs treated with cycloheximide (CHX), in the absence or presence of ConA (200 nM) for 0–150 min. d Schematic diagram of the ATF4 translational reporter, comprising the upstream open reading frames (uORF1 and uORF2) of the ATF4 5' untranslated region (5′ UTR) followed by HA-mScarlet tag replacing the ATF4 coding sequence, built on a lentiviral expression vector. The GFP control is directly driven by the cytomegalovirus (CMV) promoter. e Western blot analysis of MEFs stably expressing the ATF4 translational reporter and GFP control treated with or without Dox (30 μg/mL) or Tunicamycin (TM, 1.5 μg/mL) for 3 h, in the presence of DMSO control or ConA (200 nM). f Western blot analysis of HEK293T cells expressing control (ctrl), ATP6V0C (V0C) or ATP6V0D1 (V0D1) sgRNA, and treated with or without Dox (30 μg/mL) for 24 h. The pro and mature forms of Cathepsin B (CTSB) were as indicated. g ATF4 ChIP-qPCR analysis (n = 4 biologically independent samples) of the promoters of ATF4-targeted genes in MEFs pretreated with DMSO or ConA (200 nM) for 1 h, and then co-treated with or without Dox (30 μg/mL) for 3 h. Error bars denote SEM. Statistical analysis was performed by ANOVA followed by Tukey post hoc test (*P < 0.05; **P < 0.01; ***P < 0.001; N.S. not significant).