Figure 3.

Repression of rDNA transcription by SUMO correlates with transcriptional repression of UBF and c-Myc. A, Western blotting analysis showing the effect of ectopically overexpressed PIAS1 and/or SUMO on a panel of Pol I core transcription factors, regulators, and subunit RPA194. B, RT-qPCR analyses showing the effect of ectopically overexpressed PIAS1 and/or SUMO on the relative levels of mRNAs encoding Pol I core transcription factors and regulators. C, Western blotting analyses showing the effect of knockdown of UBC9 in HeLa cells by using either shRNA or siRNA on a panel of Pol I core transcription factors, regulators, and subunit RPA194. D, RT-qPCR analyses showing the effect of knockdown of UBC9 on the relative levels of mRNAs encoding Pol I core transcription factors and regulators. The level of each mRNA in shVec-transfected cells was set as 1. E, immunofluorescent staining assay showing that ectopically expressed PIAS1 proteins down-regulated the levels of c-Myc proteins in an E3 activity-dependent manner in HeLa cells. Also shown on the right are the rates of transfected cells with decreased levels of c-Myc proteins based on three or more representative experiments (means ± S.D.). Scale bar, 20 μm. F, immunofluorescent staining assay showing elevated levels of UBF and c-Myc proteins, but not NCL, in shUBC9-transfected HeLa cells. The rates of shUBC9-transfected cells with increased levels of UBF, NCL, or c-Myc are shown on the right, based on three representative experiments. Scale bar, 20 μm. All Western blots were performed at least three times, and the data were highly reproducible. DAPI, 4′,6′-diamino-2-phenylindole; WCL, whole cell lysate.