Figure 2
Effects of progressive fibrillarin depletion on ribosome biogenesis and p53 steady-state accumulation. (A) Fibrillarin depletion elicits an antitumoral p53-dependent nucleolar stress response. Total protein extracted from HCT116 p53 +/+ and HCT116 p53 −/− cells treated with an siRNA against fibrillarin (#528 or #529) for 1, 2, or 3 days, or with a non-targeting control siRNA (SCR, scramble) analyzed by western blotting with antibodies against fibrillarin (FBL), p53, or β-actin (loading control). Uncropped blots are shown in Supplementary Figure 8. (B) Fibrillarin expression is influenced by p53. Total protein extracted from HCT116 p53 +/+cells, from HCT116 p53 −/− cells, and from HCT116 p53 +/+cells treated with an siRNA against p53 (#210) for 3 days, analyzed by western blotting with antibodies against the indicated proteins (FBL and β-actin). Inset, p53-siRNA-mediated depletion in HCT116 p53 +/+cells is highly efficient. Total protein extracted from HCT116 p53 +/+cells treated for 3 days with an siRNA targeting p53 (#210) or with a non-targeting control siRNA (SCR). The blot was probed with antibodies against the indicated proteins (p53 and β-actin). Uncropped blots are shown in Supplementary Figure 8. (C) Fibrillarin is required for mature rRNA accumulation. HCT116 p53 +/+ and HCT116 p53 −/− cells were depleted of fibrillarin for 1, 2, or 3 days with a specific siRNA (#528 or #529) or with a non-targeting control siRNA (SCR, scramble). Total RNA was extracted and resolved on denaturing gels and mature rRNAs were visualized by ethidium bromide staining. Densitometric quantification of the signals is shown. rRNA levels in cells depleted of fibrillarin are normalized with respect to the levels observed in cells treated with a non-targeting silencer (SCR). The uncropped gel is shown in Supplementary Figure 9. Note that the effects of Fibrillarin depletion on pre-rRNA processing and p53 steady-state accumulation were observed independently at least three times, confirming for the processing data our previous observations35 (see Www.RibosomeSynthesis.Com).
