Fig. 2

PITX2 induces TGF-β signalling pathway in ovarian cancer cells. a Western blot was performed with the lysate of OAW-42 cells transiently transfected with PITX2A expression clone. b-c Q-PCR assay of TGF-B1/B2/B3 (for OAW-42 cells; b) and TGF-B2/B3 (for SKOV-3 cells; c) was done with specific primers with RNA isolated from PITX2-overexpressed respective cells. The comparative expression of respective genes is shown as relative ‘fold’ change (mean ± S.E.M). * represents p < 0.05. d-e The conditioned-medium (PITX2-CM) was collected after transient transfection with PITX2A. Freshly plated serum-starved SKOV-3 (d) and OAW-42 (e) cells were incubated for 2 h with PITX2-CM alone or in combination with 20 ng/ml TGFRI (RI) followed by Western blot of the lysates with p-SMAD2 and SMAD2 antibodies. The lysate of the cellstreated with rhTGF-β1 (rh; for 30 min) was blotted with the respective antibodies. Here, GAPDH was used as loading control. f Confocal imaging for p-SMAD2 was performed in SKOV-3 cells treated or transfected as mentioned, where the left panel shows the p-SMAD2 expression, DAPI-stained nuclei in the middle panel and the right panel shows their merged image. The images were taken at the same exposure time. Scale bar, 20 μm. g Lysates were prepared from PITX2A-siRNA trasfected OAW-42 cells for Western blot with PITX2-antibody. Here, α-tubulin was used as loading control. h OAW-42 cells were transfected with p3TP-Lux vector alone or along with expression constructs of PITX2 isoforms or pcDNA3 (empty vector) and treated with TGFRI for 16 h for luciferase assay. The activities are shown as mean fold enhancement compared to the p3TP-construct without PITX2 expression after normalization with renilla luciferase activity