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. 2012 Feb 10;31(7):1785–1797. doi: 10.1038/emboj.2012.17

Figure 6.

Figure 6

Arginine methylation and apoptosis. (A) E2F-1 arginine methylation is regulated upon DNA damage treatment. C33A or U2OS cells were treated with etoposide (Et+; 10 μM for 16 h) or left untreated (−), extracts prepared and subsequently immunoblotted with anti-E2F-1, anti-MeR–E2F-1 or actin (loading control) as indicated. (B) Apoptosis upon PRMT5 depletion. PRMT5 (P) or control (C) non-targeting siRNA (25 nM) was transfected into HCT116 p53+/+ (i), HCT116 p53−/− (ii) or U2OS (iii) cells and harvested 72 h post-transfection and analysed by FACS. The graph represents the actual percentage of sub-G1 cells in the indicated conditions in the total cell population, and the immunoblot shows the level of PRMT5 and actin control; n=3. (C) Apoptosis upon PRMT5 depletion: PRMT5 (P) or control (C) non-targeting siRNA was transfected into SAOS2 together with an expression vector encoding WT E2F-1 (1 μg) as indicated, harvested at 72 h and processed as described in (A); n=3. (D) Effect of PRMT5 siRNA in MEFs. PRMT5 (P) or control (C) non-targeting siRNA was transfected into MEFs and cells harvested at 72 h post-transfection as indicated. Cell extracts were immunoblotted with anti-PRMT5 and anti-E2F-1. Actin levels served as the loading control. (E) MEFs were treated as described in (C) and thereafter analysed by FACS. The black bar represents the effect of C siRNA, and clear bar the effect of P siRNA; n=3. (F) Apoptosis upon PRMT5 and E2F-1 depletion. PRMT5 (P), E2F-1 (E) or control (C) non-targeting siRNA was transfected into U2OS cells and harvested 72 h post-transfection and analysed by FACS as described. The graph shows the percentage of sub-G1 cells compared with the control treatment and the level of endogenous PRMT5 and E2F-1 is shown underneath; n=3. (G) Effect of PRMT5 siRNA on p73. PRMT5 (P) or control (C) siRNA was transfected into U2OS cells and cells harvested at 72 h post-transfection. Both RNA (i) and protein (ii) levels were measured, as indicated, for p73, 18S and GAPDH (i) and PRMT5, E2F-1, p73 and GAPDH (ii). (H) Regulation of apoptosis by PRMT5. Stable Tet-On cell lines expressing either WT PRMT5 or catalytically inactive ΔPRMT5 were treated with doxycycline (+; 1 μg/ml) as indicated and transfected with expression vectors encoding either WT E2F-1 or the KKK mutant derivative, or empty vector (2 μg). After 72 h, cells were harvested and analysed by FACS. The graph represents the actual percentage of sub-G1 cells in the indicated conditions (each treatment in triplicate); n=3. (I) The immunoblot shows the level of PRMT5 and E2F-1 for the experiment described above (H). Actin levels served as a loading control. Figure source data can be found with the Supplementary data