Figure 1.
Knockdown of PRMT6 results in upregulation of p21.
(A) U2OS cells were transfected with the indicated siRNAs. After 72 hours cells were harvested and immunoblot analysis was performed using antisera against PRMT6, PRMT1, p21,p27 and p57. (B) MCF7 (right panel) or HCT116 (left panel) cells were transfected with the indicated siRNAs. After 72 hours cells were harvested and immunoblot analysis was performed using antisera against PRMT6, p21, p27, p57 and GAPDH. (C) U2OS cells were transfected with expression constructs for pcDNA3-PRMT6 wild type and pcDNA3-PRMT6-E164Q or untreated (mock). After 48 hours cells were harvested and analysed with the indicated antisera.
Figure 2.
Transcriptional regulation of p21 and p27 by PRMT6.
(A) U2OS cells were transfected with the indicated siRNAs and harvested 72 h later. RT-qPCR was performed with primers for p21, p27 and 57. Gene expression was normalized for beta-Actin. Error bars indicate the standard error of the mean. (B) Cells were transfected with p21-Luc reporter and the indicated siRNAs. Relative light units represent the mean value of a triplicate transfection.
Figure 3.
PRMT6 regulates p21 and p27 on the promoter level.
(A) ChIP analysis was done 46 h upon transient expression of mycPRMT6 using primers for the transcriptional start site (TSS) and a control region of the p21 promoter. (B) ChIP analysis as in A, using promoter specific primers for the TSS of p27 and p57.
Figure 4.
Knockdown of PRMT6 results in an accumulation of cells in G2.
(A) U2OS cells were transfected with the indicated siRNAs and fixed 72 h later. For DNA content analysis, cells were stained with propidium iodide and analyzed by flow cytometry. Percentage of cells with a 4n DNA content is indicated. This is a representative example of three biological replicates. (B) Same as in A, this time cells were double stained using propidium iodide and Phospho-H3S10 antibody. Percentage of mitotic cells is indicated.
Table 1.
Cell cycle distribution of control and siPRMT6 transfected U2OS cells.