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. 2019 Aug 20;11(8):1211. doi: 10.3390/cancers11081211

Figure 2.

Figure 2

Effect of PAR2 knockdown on RAC1B knockdown-induced ALK5 expression in Panc1 cells. (A) Panc1 cells were transfected twice with 25 nM each of Co siRNAs specific for RAC1B and PAR2, 25 nM each of RAC1B siRNA + Co siRNA specific for PAR2, or 25 nM each of RAC1B siRNA + PAR2 siRNA. Forty-eight hours after the second transfection, these Panc1-RAC1B-KD cells were subjected to RNA isolation and qPCR analysis for ALK5. Data represent the normalized mean ± SD from three independent experiments and are displayed relative to control siRNA-transfected cells set arbitrarily at 1.0. (B) As in (A) except that RAC1B-knockdown cells were analyzed by immunoblotting for ALK5, RAC1B, and HSP90. The graph below the immunoblot shows results from densitometry-based quantitative analysis of ALK5. Data represent the mean ± SD from three experiments. (C) As in (A), except that the indicated transfectants were treated, or not, with TGF-β1 for 24 h prior to qPCR analysis of ALK5. (D) As in (A), except that cells were cotransfected with ALK5 siRNA instead of PAR2 siRNA and subjected to qPCR analysis of PAR2. (E) Panc1-RAC1B-KO cells (KO), and vector control cells (V) were transfected twice with 50 nM each of either Co siRNA or PAR2 siRNA. Forty-eight hours after the second transfection, the cells were subjected to immunoblot analysis of ALK5 and GAPDH. The asterisks in (A–C,E) indicate significance. The thin vertical lines in the immunoblot images of panels B and E indicate that irrelevant lanes have been removed.