Fig. 6: GPER KO in MDA-MB-231 cells suppresses the CTGF-driven cytoskeleton reorganization and migration in CAFs. | Cell Death Discovery

Fig. 6: GPER KO in MDA-MB-231 cells suppresses the CTGF-driven cytoskeleton reorganization and migration in CAFs.

From: GPER deletion triggers inhibitory effects in triple negative breast cancer (TNBC) cells through the JNK/c-Jun/p53/Noxa transduction pathway

Fig. 6

mRNA (A) and protein (B) levels of CTGF evaluated respectively by real-time PCR and immunoblotting in WT and GPER KO MDA-MB-231 cells. In RNA experiments, values are normalized to the actin beta (ACTB) expression and presented as fold changes of mRNA expression upon GPER KO relative to WT. C Evaluation by immunoblotting of CTGF protein levels in conditioned medium (CM) collected from WT and GPER KO MDA-MB-231 cells. Ponceau red staining of the membrane was used as a loading control for the CM. Side panels show densitometric analysis of the blots normalized to GAPDH that served as loading control. D FITC-phalloidin staining of CAFs cultured for 12 h with conditioned medium (CM) collected from WT and GPER KO MDA-MB-231 cells, which were previously treated for 24 h with vehicle or 100 nM CTGF, as indicated. Cells were stained with FITC-phalloidin to detect F-actin stress fibers (green) and with DAPI to detect nuclei (blue). The images shown represent 10 random fields from three independent experiments. E Corrected total cell fluorescence (CTCF) was calculated on at least 10 pictures from each sample. Scale bar: 75 μm. Results shown are representative of at least three independent experiments. Values represent the mean ± SD of three independent experiments performed in triplicate. F Transwell migration assay in CAFs cultured for 12 h with conditioned medium (CM) collected from WT and GPER KO MDA-MB-231 cells, which were previously treated for 24 h with vehicle or 100 nM CTGF, as indicated. G Evaluation of cell migration in 10 random fields in each of three independent experiments performed in triplicate. Scale bar: 200 μm. (*) and (■) indicate p < 0.05. (***) indicate (p < 0.001). H Cartoon depicting the effects of GPER KO (1) in inhibiting the expression of CTGF (2) and its secretion (3) by MDA-MB-231 cells, thus preventing F-actin cytoskeleton reorganization and migratory effects (4) in CAFs. Created with BioRender.com.

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