Figure 2.

HRI controls proteotoxicity in the face of proteasome inhibition through modulation of autophagy.A–D, HEK293T cells transduced with lentiviral particles targeting a scrambled sequence (SC) and HRI (shHRI) were treated for 4 h with DMSO (control), 10 μM MG132, and 10 nM bafilomycin, and cell extracts were collected for Western blot analysis. Western blotting was performed using antibodies against phospho-eIF2α (ser 51) or eIF2α (A), ATF4 (B), LC3 A/B or p62 (C), and cleaved PARP-1 (D). GAPDH and Tubulin were used as loading controls. E, scrambled (SC) sequence and HRI (shHRI) HeLa cells treated either with DMSO (CTR) or with 5 μM MG132 for 4 h was followed by fixation with ice-cold 100% methanol for 5 min. Subsequently, immunofluorescence analysis was executed using anti-p62 and anti-LC3 antibodies. A–D and E are representative of four and three independent experiments, respectively. The scale bar represents 10 μm.