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

Figure 4.

Figure 4.

rDNA transcription is not repressed by endogenous SUMO in cells deficient in CDK9 SUMOylation. A, characterization of CDK9WT and CDK9K/R mutant cell lines. These HEK293T cell lines, with both endogenous CDK9 genes disrupted by CRISPR-Cas9, expressed either Myc-tagged WT CDK9 (CDK9WT) or a mutant CDK9 with all lysine residues converted to arginines (CDK9K/R). B, RT-qPCR analysis showing the effect of knockdown of UBC9 by shRNAs on the relative levels of UBF, c-Myc, and NCL mRNAs in both CDK9WT and CDK9K/R cells. Note that the level of mRNA for each protein in shVec-transfected cells was set as 1. C, RT-qPCR analysis showing the effect of knockdown of UBC9 by siRNA on the relative levels of UBF, c-Myc, and NCL mRNAs in both CDK9WT and CDK9K/R cells. Note that the level of mRNA for each protein in scramble control siRNA-transfected cells was set as 1. D, Western blotting analysis showing the effect of knockdown of UBC9 by shRNAs on the levels of c-Myc, UBF, and NCL proteins in both CDK9WT and CDK9K/R cells. E, Western blotting analyses showing the effect of knockdown of UBC9 by siRNA on the levels of c-Myc, UBF, and NCL in both CDK9WT and CDK9K/R cells. F, Western blotting and RT-qPCR analyses showing that knockdown of UBC9 by shRNAs led to elevated Pol I transcription in CDK9WT but not in CDK9K/R cells. Note that the level of pre-RNA in shVec-transfected cells was set as 1. G, Western blotting and RT-qPCR analyses showing that knockdown of UBC9 by siRNAs led to elevated Pol I transcription in CDK9WT but not in CDK9K/R cells. Note the level of pre-RNA in control scramble siRNA-transfected cells was set as 1. H, the EU incorporation assay showing that knockdown of UBC9 by transfected shRNAs elevated the level of rDNA transcription in CDK9WT but not in CDK9K/R cells. Transfected cells are marked by arrowheads. The rates of cells with increased EU incorporation versus counted transfected cells are shown on the right, based on three representative experiments. Scale bar, 20 μm. DAPI, 4′,6′-diamino-2-phenylindole.