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. 2019 Nov 6;294(50):19155–19166. doi: 10.1074/jbc.RA119.010624

Figure 2.

Figure 2.

Multiple PIAS family proteins are involved in repression of rDNA transcription. A, effect of knockdown of PIAS family proteins on rDNA transcription. HeLa cells were transfected with siRNA against each of the PIAS family proteins, and 2 days after transfection the relative levels of 47S rRNA precursor were measured by RT-qPCR. B, effect of knockdown of PIAS1 and PIAS4 by specific shRNAs on rDNA transcription. HeLa cells were transfected with either control shVec or two different PIAS1 or PIAS4-specific shRNAs. Two days after transfection, the relative levels of 47S rRNA precursor were measured by RT-qPCR. C, the EU incorporation assay showing that knockdown of PIAS1 or PIAS4 in HeLa cells all led to increased rRNA synthesis. The shRNA-transfected cells are marked by arrowheads. Also shown on the right are the rates of transfected cells with increased levels of EU incorporation counted from three or more representative experiments (means ± S.D.). D, the EU incorporation assay showing the effect of ectopic overexpression of various PIAS proteins with or without SUMO1 on rDNA transcription in HeLa cells. Transfected cells are marked by arrowheads. Note that PIAS1, PIAS2b, and PIAS4 were able to repress rDNA transcription when they were ectopically overexpressed. Also shown on the right are the rates of transfected cells with decreased levels of EU incorporation counted from three or more representative experiments (means ± S.D.). Scale bar, 20 μm. E, the EU incorporation assay showing that ectopically expressed PIAS1 repressed rDNA transcription in HeLa cells in an E3 ligase activity-dependent manner. Transfected cells are marked by arrowheads. Also shown on the right are the rates of transfected cells with decreased levels of EU incorporation counted from three or more representative experiments (means ± S.D.). Scale bar, 20 μm. F, RT-qPCR analyses showing that ectopically expressed PIAS1 repressed rDNA transcription in HeLa cells in an E3 activity-dependent manner, and the repression was augmented by co-expressed SUMO1. DAPI, 4′,6′-diamino-2-phenylindole.