Research Paper Volume 12, Issue 3 pp 2507—2529

Figure 3. PRMT5 inhibits DNA damaging agents-induced OS cell senescence. (A and B) Cisplatin (CDDP, 10 μM) was added to SC, shP5#1, and shP5#3 U2 OS cells for 24 h. Then, DSBs were visualized by a comet assay, followed by quantification of the OTM with Open Comet software. Scale bar = 20 μm. **, p< 0.01. (C) SC, shP5#1 and shP5#3 U2 OS cells were treated with 10 μM CDDP for 24 h, the expressions of PRMT5 and γ-H2A.X were measured by WB. (D, E) SC, shP5#1 and shP5#3 U2 OS cells were treated with 20 μM CDDP for 3 h; the medium was then replaced with fresh medium, and cells were cultured for 12 h or 24 h (time for DNA repair). Antibody against γ-H2A.X was used for immunofluorescence staining, DAPI was used to counterstain the nucleus, and the percentage of positive cells (with ≥10 foci per nucleus considered positive) was counted in three independent experiments and quantified with ImageJ software. Scale bar = 10 μm. *, p< 0.05. (F) SC, shP5#1 and shP5#3 U2 OS cells were treated with 10 μM CDDP for 24 h, the expressions of PRMT5, p16, p21 and p53 were measured by WB. (G, H) SC, shP5#1 and shP5#3 U2 OS cells were treated with 10 μM CDDP for 12 h, the percentage of senescent cells was quantified. *, p< 0.05; ***, p< 0.001; the cellular senescence was visualized using a SA-β-gal staining kit. Scale bar = 50 μm. (I, J) U2 OS cells were transfected with plasmids encoding HA-PRMT5, followed by treated with CDDP for 12 h, and the percentage of senescent cells was quantified. ****, p< 0.0001; the expressions of PRMT5, TXNIP and p21 were determined by WB; the cellular senescence was visualized using a SA-β-gal staining kit. Scale bar = 50 μm.