Fig. 3: Pre-rRNA accumulation is an adaptive response to nutrient restoration.
From: Glutamine deficiency in solid tumor cells confers resistance to ribosomal RNA synthesis inhibitors
a Pre-rRNA expression after treatment with vehicle (NaH2PO4), CX-5461 (10 µM), or BMH-21 (1 µM) under 24 h ND. Data represent mean ± SD of n = 3 technical replicates. P values (left to right): HCT116, <0.0001, <0.0001, A375, <0.0001, <0.0001, statistical analysis by two-way ANOVA. b Percentage of cell death in HCT116 cells cultured in control or ND medium with vehicle (NaH2PO4), CX-5461 (10 µM), or BMH-21 (1 µM) as assessed using trypan blue exclusion assay. Data represent mean ± SD of n = 3 biological replicates. (ns: not significant). P values (left to right): ND, <0.0001, <0.0001, statistical analysis by two-way ANOVA. c Schematic showing ND and nutrient restoration (NR) treatments. To inhibit pre-rRNA expression, HCT116 or A375 cells were first placed under ND in the presence of vehicle, CX-5461, or BMH-21. Cells pretreated to ND + CX-5461 (10 µM)/ND + BMH-21 (1 µM) represent “pre-rRNA-depleted” cells. To induce metabolic recovery, pre-starved cells were washed three times with PBS and were allowed to rest in drug-free DMEM (10% FBS). d In order to assess the expression of ribosome-free proteins, input lysates were clarified of large ribosomes using sucrose cushion ultracentrifugation. The resulting ribosome-free lysates were analyzed using western blotting. e Expression of ribosome-free r-proteins in metabolically recovering HCT116 cells. f NR induces cell death in pre-rRNA-depleted HCT116 and A375 cells. Representative microscopy images were taken after 24 h of NR. Scale bar, 100 μm. g Percentage of cell death of metabolically recovering cells as determined using trypan blue exclusion assays. Data represent mean ± SD of n = 3 biological replicates. P-values (left to right): HCT116, <0.0001, <0.0001, A375, <0.0001, <0.0001, statistical analysis by two-way ANOVA. h NR induces apoptosis in pre-rRNA-depleted cells, which is inhibited by the pan-caspase inhibitor zVAD-FMK (1 µM). i Pre-rRNA synthesis resumes after CX-5461 (10 µM)/BMH-21 (1 µM) drug washout and NR. Data represent mean ± SD of n = 3 independent images. P values (left to right): 0.077, 0.0422, statistical analysis by two-way ANOVA. Scale bar, 10 μm. For a, b, g, i, ns, not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.
