Fig. 2: Pre-rRNA processing is impaired under ND.
From: Glutamine deficiency in solid tumor cells confers resistance to ribosomal RNA synthesis inhibitors
a Analysis of pre-rRNA processing kinetics in control or ND HCT116 cells. HCT116 cells that were pretreated to 3.5 h ND were subsequently pulse labeled with L-(methyl-3H)-methionine for 30 min and chased with excess cold methionine. To assess the effect of nutrient restoration on pre-rRNAs accumulated under ND, Gln was added back to the ND medium 2 h after the cold methionine chase (“ND + Gln”). b Quantification of the 28S/18S bands from Fig. 1f (as indicated by the red triangles). The black arrow at the 2 h time point indicates the time at which glutamine was added to starved (ND) cells. c ND increases the pre-rRNA half-life (λ) as assessed using qRT-PCR. HCT116 cells were pretreated to (24 h) ND, transcription was stopped by (20 µM) actinomycin D, and total RNA at the indicated time points for qRT-PCR analysis. Data represent mean ± SD of n = 3 technical replicates. P values (left to right): 0.006, 0.0007, 3.2 × 10−6 (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001). d ND slows pre-rRNA processing in A375, A549, U2OS, MKN45 cells. Data represent mean ± SD of n = 3 technical replicates. P values (left to right): A375, 0.00081, 7.2 × 10−6, A549, 0.0035, 9.4 × 10−5, U2OS, 0.0015, 0.0031, MKN45, 0.00025, 0.00031 (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001). e HCT116 tumor pre-rRNA half-life as determined using qRT-PCR. Data represent mean ± SD of n = 3 technical replicates. f Expression of pre-rRNAs, 18S, and 28S in HCT116 tumors after actinomycin D (1.33 mM) intratumoral injection. Data represent mean ± SD of n = 4 technical replicates. P values (left to right): 6.5 × 10−5, 0.20, 0.12 (ns, not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001). g Schematic of core and periphery regions of established HCT116 tumors (approximate size 1000 mm3). h Expression of HIF-1α in HCT116 periphery and core tissues as assessed using western blotting. i Pre-rRNA processing is faster in peripheral tissues compared to core tissues, as determined using qRT-PCR. Data represent mean ± SD of n = 3 technical replicates. P values (left to right): 0.046, 0.00064, 0.00016 (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001).
