Fig. 1: ISRIB aggravates UPS impairment during proteotoxic stress.

a Representative micrographs of the effect of ISRIB on Ub-YFP in thermally stressed cells. Cells were left untreated (-ISRIB) or pretreated with ISRIB (+ISRIB) for 30 min. After this initial incubation, they were left untreated (−HS) or exposed to 43 °C for 30 min (+HS) and followed for 4 h after the heat shock in the absence of ISRIB (HS recovery). Images were captured with a wide-field, high-content microscope. Scale bar, 20 μm. b Quantification of mean cellular YFP fluorescence intensities per cell. Experimental setup as described in (a). Values were normalized to untreated control cells (-ISRIB, −HS). The frequency and distribution of the relative fluorescence intensities per cell are shown as violin plots. The solid lines in each distribution represent the medians, and the dash lines represent the upper and lower interquartile range limits (three independent experiments, >1000 cells analyzed per condition, Kruskal–Wallis test, **P < 0.01, n.s.: not significant). c Effect of ISRIB on accumulation kinetics of Ub-YFP. Kinetics of Ub-YFP accumulation in control (-ISRIB) and ISRIB-treated (+ISRIB) MelJuSo cells, in response to a 30-min 43 °C heat shock, followed by 14 h at 37 °C (HS recovery). Data points show the mean fluorescence intensity per image. Four sites were imaged, per treatment, per time point. Error bars indicate the standard deviation between the four images taken for the same condition (time and treatment). Here we show one representative experiment out of two independent replicates. d Effect of ISRIB on Ub-YFP accumulation in stressed cells. MelJuSo cells expressing Ub-YFP were incubated in the absence (-ISRIB) or presence of ISRIB (+ISRIB) for 30 min, before being subjected to 43 °C heat shock for 30 min. The Ub-YFP levels were followed throughout the recovery phase (HS recovery). e Pulse-chase experiment with HPG-labeled Ub-YFP. The upper panel is an in-gel detection of HPG labeled Ub-YFP in a pulse-chase experiment during the recovery phase, in the absence (-ISRIB) or presence of ISRIB (+ISRIB). The total Ub-YFP reporter levels were detected in the lysate by immunoblotting (lower panel). A representative result of three independent experiments is shown. f Effect of ISRIB on ubiquitin-independent proteasomal degradation. Quantification of GFP intensities of MelJuSo cells expressing GFP-ODC. Cells were left untreated (-ISRIB) or incubated with ISRIB (+ISRIB) for 30 min before being left untreated (37 °C) or exposed to a 43 °C heat shock for 30 min, followed by recovery (HS recovery) for 4 h. Values were normalized to untreated control cells (-ISRIB, −HS). The violin plots represent the frequency and distribution of the relative fluorescence intensities per cell, quantified as nuclear YFP intensity.