Fig 1: Expression of most ribosomal protein genes is integral for maintaining cellular p53 homeostasisGiven the enrichment of ribosomal protein (RP) genes in our primary screen dataset, we further investigated this group; depicted is the breakdown of screened RPs that were p53 “positive,” 2-fold or greater increase in p53, and the proportion of which are in the large (60S) or small (40S) ribosome subunit, shown in (A). We further verified the p53 result of approximately 50% of the RP genes (when depleted using siRNAs for 72 h) with quantitative p53 analysis (Alphascreen) in A549 cells (note genes associated with DBA are highlighted in red) (B). We selected candidates that were “p53 positive” (RPS18, RPL21, RPS19) and “p53 low” (RPL5, RPL11, RPL22, RPL28) to confirm knockdown at the protein level, and we determined p53 and p21 protein levels using western blot analysis in A549 cells (C, representative blot of n = 3 experiments). Cells depleted of each RP were then subjected to ribosome subunit analysis (performed under high-salt conditions, D) to determine the effect of depletion on 60S and 40S subunits (quantitation of 40S:60S subunit ratio is presented in E). Comparison of the timing of RP incorporation into the ribosome subunit as tabulated by de la Cruz and colleagues (de la Cruz et al., 2015) with p53 intensity when the RP was depleted using siRNA (F), where red circles indicate 40S subunit RPs, and black circles indicate 60S subunit RPs. Quantitative data presented as mean -/+ SD. Statistical analysis: for quantitation of 40S:60S subunits, one-way ANOVA with Dunnett’s multiple comparison test was conducted; for timing of RP incorporation into ribosome subunit, one-way ANOVA with Tukey’s multiple comparison test was performed. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns = not significant. Alphascreen analysis performed n = 3–5 biological experiments; ribosome subunit analysis conducted on a minimum of n = 3 biological experiments per candidate.
Supplier Page from Abcam for Anti-RPL28 antibody