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The Indolinone MAZ51 Induces Cell Rounding and G2/M Cell Cycle Arrest in Glioma Cells without the Inhibition of VEGFR-3 Phosphorylation: Involvement of the RhoA and Akt/GSK3β Signaling Pathways

Figure 4

The effects of MAZ51 in C6 cells are independent of the inhibition of VEGFR-3 phosphorylation.

(A) C6 cells were treated with 2.5 µM or 5.0 µM MAZ51 for 6 h or 24 h. In parallel, cells were treated with recombinant rat VEGF-C protein (150 ng/mL) for 20 min. Cell lysates were immunoprecipitated with anti-VEGFR-3 antibody and subsequently assayed by western blot analysis using an anti-phosphotyrosine antibody. The loading control is represented by the western blot of VEGFR-3 with the double bands indicating the larger glycosylated (195 kD) and smaller cleaved (125 kD) forms. Effects of treatment with VEGF-C on cytoskeletal rearrangements (B) and cell cycle distribution (C) of C6 cells. C6 cells were treated with VEGF-C (150 ng/mL) for 20 min, while control cells were treated with 0.1% DMSO. (D) Transient transfection using VEGFR-3 siRNA or scrambled RNA (mock) for the indicated time periods. C6 cells transfected with VEGFR-3 siRNA exhibited the most extensive knockdown of the protein level at 72 h after transfection. (E) Effects of 5 µM MAZ51 on the morphological and cytoskeletal rearrangements of C6 cells after transfection with VEGFR-3 siRNA or scrambled siRNA. Data are representative of three independent experiments. Scale bar = 50 µm.

Figure 4