Fig. 5: ISRIB impairs degradation of ribosome quality control an RQC reporter substrate.

a Schematic representation of the readthrough double reporters GFP-P2A-K0-P2A-RFP (K0) and GFP-P2A-K20-P2A-RFP (K20). b Flow cytometric quantification of MelJuSo cells transfected with pmGFP-P2A-K20-P2A-RFP (K0) or pmGFP-P2A-K20-P2A-RFP (K20) respectively. Cells expressing GFP-K20-RFP were either pre-treated in the absence (-ISRIB) or presence (+ISRIB) of ISRIB for 30 min. After pre-treatment, cells were left untreated (−HS), or subjected to 43 °C heat shock (+HS) followed by 4 h recovery (HS recovery). The readthrough region was gated in K0 transfected cells, exhibiting linear correlation between GFP and RFP fluorescence. Data represent the mean ± SD. (three independent experiments, n.s.: not significant). c Effect of ISRIB on RQC substrate degradation. Representative micrographs of MelJuSo cells expressing GFP^nonstop. Cells were left untreated (-ISRIB), incubated with ISRIB for 30 min (+ISRIB), incubated with proteasome inhibitor epoxomicin for 4 h (+EPX) or combined ISRIB with proteasome inhibitor epoxomicin (ISRIB + EPX) before being left untreated (−HS) or exposed to a 43 °C heat shock for 30 min and followed by recovery (HS recovery) for 4 h. Images were captured with wide-field, high-content microscope. Scale bar, 20 μm. d Quantification of mean cellular YFP fluorescence intensities in MelJuSo cells stably expressing GFP^nonstop. Cells were pre-treated with or without ISRIB for 30 min and either left untreated (−HS) or exposed to a 43 °C heat shock for 30 min followed by 4 h (HS recovery), in presence or absence of the proteasome inhibitor epoxomicin (+EPX). 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. (three independent experiments, >1000 cells analyzed per condition, Kruskal–Wallis test, **P < 0.01, n.s.: not significant).