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. 2016 Jul 30;7(34):55458–55472. doi: 10.18632/oncotarget.10969

Figure 4. ROCK/Rho pathway is activated by TBXA2R.

Figure 4

(A) Western blot analysis of total and phosphorylated ERK1/2 in MDA-MB-231 and (B) total and phosphorylated AKT (Ser 473) levels in SUM-PT-149 cells 72 hr post-transfection of 2 independent TBXA2R siRNAs. Densitometry values were normalised firstly for total ERK to β-tubulin and then phosphorylated values were normalised against total ERK/β-tubulin. (C) Western blot analysis of total and phosphorylated MLC2 (Ser 19) levels in SUM-PT-159 and MDA-MB-231 cells 72 hr post-transfection with 2 independent TBXA2R siRNAs with accompanying graphs showing densitometry values for phospho-MLC2 (Ser 19) relative to total MLC2 levels in SUM-PT-159 cells. (D) Western blot analysis of GTP-RhoA and GTP-RhoC levels were performed in the same two TNBC cell lines 72 hr post-transfection with 2 independent TBXA2R siRNAs, with accompanying graphs showing densitometry values for active Rho relative to total Rho levels. GAPDH was used as a loading control in (A–D) and blots were quantified by densitometry of 3 independent replicates. (E) Graphs showing absorbance values (n = 3) for crystal violet staining of MDA-MB-231 and (F) hTERT HME-1 cells 72 hr post-transfection with RhoA, RhoC, ROCK1 and ROCK2 siRNAs with representative images of plates underneath each graph. Data was analysed by one-way ANOVA with Dunnett's post-hoc test (A, B, D) or unpaired two-tailed t-test (C, E, F) ; **P < 0.01.