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. 2014 Oct 7;42(19):11928–11940. doi: 10.1093/nar/gku929

Figure 2.

Figure 2.

Phospho-ETS1 is required for the migration of the RAS-active prostate cancer line, DU145. (A) Immunoblot with antibodies shown (left) of DU145 cells with shRNA mediated knockdown of five ETS factors (top). An shRNA targeting luciferase is a negative control. Tubulin is a loading control. (B) A transwell assay measured relative cell migration of DU145 cells with indicated knockdown. Mean and SEM of ≥ 3 biological replicates shown. P-values (*<0.05, **<0.01, ***<0.001) are calculated by two-tailed T-test. (C) An MTT assay compared proliferation of DU145 cells with ETS1 and control shRNA knockdowns. Three biological replicates shown. Proliferation rates are not significantly different (P = 0.73). (D) A reporter assay compares relative luciferase units (firefly/renilla) from DU145 cells expressing control luciferase (luc) shRNAs or ETS1 shRNAs and treated with the MEK inhibitor, U0126 50 μM, as indicated. The firefly luciferase vector has three copies of the ETS/AP1 element (WT) 5′ to the minimal promoter or the same vector with point mutations in each ETS binding site (MUT). Values are shown as a ratio to the first column and are the mean and SEM of three biological replicates. P-values are as in (B). (E) Anchorage-independent growth by soft agar assay for selected ETS knockdown in DU145 cells. Number of colonies calculated relative to luciferase shRNA. Mean and SEM of three biological replicates reported. (F) Immunoblots show levels of E-cadherin and Vimentin in DU145 cells with the indicated shRNA knockdown. (G) Immunoblot with antibodies shown (left) for DU145 cells transduced with a retrovirus to stably overexpress empty vector, Flag-ETS1, Flag-T38A/S41A ETS1 and Flag-ETS2. (H) A transwell assay measured relative cell migration of DU145 cells with indicated ETS overexpression constructs as in (G). Mean and SEM of ≥3 biological replicates each containing two technical replicates are shown. P-value of ETS1 compared to T38A/S41A ETS1 by T-test. (I) Relative luciferase expression as in (D) for a 3xETS/AP-1 reporter in DU145 cells overexpressing indicated ETS construct.