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. 2012 Mar 16;19(9):1514–1524. doi: 10.1038/cdd.2012.28

Figure 4.

Figure 4

Regulated actin-severing function of villin is required for its anti-apoptotic function. (a) Villin is tyrosine-phosphorylated in CPT-treated MDCK cells. Tyrosine-phosphorylated villin was identified by immunoprecipitation (IP) of villin and immunoblot (IB) analysis with phosphotyrosine antibody (PY-20) and vice versa. The immunoblot was re-probed with anti-villin antibody. Data are representative of four experiments with similar results. (b) Inhibiting tyrosine phosphorylation of villin with the Src kinase inhibitor PP2 (10 μM) reverses the anti-apoptotic function of villin. The negative control PP3 (10 μM) had no effect on apoptosis in VIL/WT cells). Apoptotic cells were visualized by phase-contrast microscopy and identified using Hoechst 33258 staining. All experiments were performed in triplicate, with similar results. Bar, 50 μm. PI positive cells were identified by flow cytometry. There was a significant increase (*P<0.01, n=9) in PI positive VIL/WT pre-treated with the Src kinase inhibitor PP2. (c) MDCK Tet-Off cells expressing the phosphorylation site mutant of villin, VIL/AYFM failed to inhibit CPT-induced apoptosis. Apoptotic cells were visualized in VIL/WT and VIL/AYFM cells by phase-contrast microscopy and identified using Hoechst 33258 staining. All experiments were performed in triplicate, with similar results, n=6. Bar, 50 μm. PI positive cells were identified by flow cytometry. There was a significant increase in the number of PI positive VIL/AYFM cells in response to CPT treatment (*P<0.01, n=9). (d) Caspase-3 activity was significantly inhibited in VIL/NULL and VIL/AYFM cells between 4–6 hours post-treatment (*AYFM compared with VIL/WT cells; #NULL cells compared with VIL/WT; P<0.01, n=6)