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. 2016 Sep 5;44(20):9803–9820. doi: 10.1093/nar/gkw790

Figure 1.

Figure 1.

Depletion of AATF impairs nucleolar steps of 40S ribosomal subunit biogenesis. (A) Indicated proteins were depleted by RNAi for 48 h in HeLa cell lines expressing RPS2-YFP or RPL29-GFP in an inducible manner. Expression of RPS2-YFP and RPL29-GFP was induced with tetracycline (125 ng/ml) for 16 h and subsequently chased for 4 h by washing out the tetracycline. Note that the strong nucleolar signal of RPL29-GFP in control cells is due to the slow incorporation of the reporter protein into nucleolar pre-ribosomal particles. Cells were fixed and analyzed by confocal microscopy. Scale bar, 20 μm. (B) siRNA treatment in HeLa cells for 72 h. Localization of ENP1/BYSL was analyzed by immunofluorescence (IF). To visualize early defects in ribosome maturation, CRM1-mediated export of 40S ribosomal subunits was blocked by Leptomycin B treatment (LMB, 20 nM, 2 h). (C) RNAi efficiency was verified by Western blot analysis after 72 h.