Extended Data Fig. 4: NADPH collapse does not cause glucose starvation-induced cell death.
From: The atypical E3 ligase HOIL-1 safeguards the ribosome during cellular stress
a, Heatmap of metabolite levels after 24 h glucose starvation represented as log2(FC) of parental AC16 cells. b, Pathway analysis of metabolites enriched in HOIL-1ΔRBR cells after 24 h glucose starvation. c, Cell death in AC16 cells after DMSO vehicle, uridine (25 mM), hydroxyurea (0.2 mM, 2.0 mM), brequinar (0.1 µM, 1.0 µM), or oxythiamine (0.5 mM, 5 mM) treatment concurrent with 48 h glucose starvation (n = 3 biological replicates). d, Cell death in AC16 cells stably overexpressing (OE) xCT treated with erastin (2 µM) concurrent with 48 h glucose starvation (n = 3 biological replicates for all except parent-xCTOE + erastin, n = 2 biological replicates). e, Cell death in AC16 grown in L-cystine and glucose-free media supplemented with L-cystine (200 µM) or sulfasalazine (500 µM) for 48 h (n = 4 biological replicates). f, Schematic of NADP + and/NADPH flux. g, Measurement of NADP+ and NADPH ratio during glucose starvation in xCTKO AC16 cells (n = 3 biological replicates). h, Measurement of NADP+ and NADPH ratio in AC16 cells treated with G6PD inhibitor (G6PDi-1, 50 µM) during glucose starvation (n = 3 biological replicates). i, Cell death in AC16 cells after DMSO vehicle, G6PDi-1 (50 µM), or 6-aminonicotinamide (6-AN, 20 µM) treatment concurrent with 48 h glucose starvation (n = 3 biological replicates). j, Cell death in AC16 cells stably overexpressing doxycycline (dox)-inducible TPNOX treated with dox (1 µg/ml) for either 24 h prior to (pre-treat) or concurrently (co-treat) with 48 h glucose starvation (n = 3 biological replicates). k, xCT-related metabolite levels at 24 h glucose starvation as measured by untargeted LC-MS represented as fold change over parent (n = 6 biological replicates). Data are presented as mean ± s.e.m from one representative experiment (c-e, g-k). P values were determined by two-way ANOVA followed by Tukey’s (e) or Dunnett’s (g, k) post-hoc comparison. Source numerical data are available in Source Data.
