Fig. 5: Loss of the HOIL-1 RBR domain remodels the ribosome ubiquitin landscape and renders glucose starvation ribotoxic.

a, A schematic of the experimental design. b, A volcano plot showing differentially ubiquitinated peptides after 8 h glucose starvation. Significant at −log(FDR) ≥2s.d. ≤−2s.d. c, Gene Ontology enrichment analysis of proteins with reduced ubiquitin-modified peptides in HOIL-1^ΔRBR cells relative to parent AC16 cells after 8 h glucose starvation. d, A schematic of RSR signalling. e, An immunoblot analysis of AC16 cells after DMSO vehicle, TNF (10 ng ml⁻¹), anisomycin (5 µM) or glucose starvation for the indicated timepoints. ZAKα phosphorylation was assessed using a Phos-Tag acrylamide gel. f, An immunoblot analysis of AC16 cells after glucose starvation for the indicated timepoints. g, An immunoblot analysis of AC16 cells transfected with ZAK or NTC siRNA 48 h before sodium arsenite (10 µM) or glucose starvation for the indicated timepoints. h, Cell death in AC16 cells transfected with ZAK or NTC siRNA before glucose starvation for 48 h (n = 4 biological replicates). i, Cell death in 786-O cells transfected with ZAK or NTC siRNA before glucose starvation for 48 h (n = 4 biological replicates). j, Immunoblot analysis of 786-O cells transfected with ZAK or NTC siRNA before glucose starvation. Data are presented as mean ± s.e.m. from one representative experiment. FDR values were calculated using moderated t-tests in the limma R package followed by the Benjamini–Hochberg procedure (b); P values were determined using the hypergeometric test in ShinyGO (c) or two-way ANOVA followed by Holm–Šídák’s post hoc comparison (h and i).

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