Skip to main content
. 2016 May 27;7(27):41095–41109. doi: 10.18632/oncotarget.9600

Figure 2. Effects of PAR2 depletion on the TGF-β1 response of invasion associated genes.

Figure 2

A. Panc-1, Colo357, and HaCaT cells were transfected twice on two consecutive days with 50 nM of control (Co), PAR2, PAR1 or ALK5 siRNA, as indicated, using Lipofectamine RNAiMAX. Twenty-four h after the second round of transfection, cells were stimulated, or not, with 5 ng/ml TGF-β1 for another 24 h (Panc-1, Colo357) or 48 h (HaCaT) followed by RNA isolation and qPCR for the indicated genes. B. Panc-1 cells were transfected as described in A using the indicated siRNAs. Twenty-four h after the second round of transfection, cells were stimulated, or not, with 5 ng/ml TGF-β1 for 1 h followed by RNA isolation and qPCR for GADD45β and Smad7. In A and B, bars represent mean values ± SD of three wells normalised to β-actin and TBP. Successful knockdown of PAR2, ALK5 and PAR1 expression was verified by qPCR (not shown). One representative experiment out of three experiments performed in total is shown. P < 0.05 relative to the respective TGF-β1-treated Co. C. Primary aortic smooth muscle cells were isolated from PAR2−/− and strain-matched (C57/Bl6J) control wild-type mice and cultured in DMEM with 10% FBS. Prior to stimulation, the aortic smooth muscle cells were seeded in 6-well plates at about 70% confluence. After 24 h of serum withdrawal, cells were incubated with 5 ng/ml TGF-β1 for the times indicated and subsequently processed for RNA isolation and qPCR analysis for PAI-1 and GAPDH. Data shown represent mean values ± SD after normalisation for GAPDH from eight mice per group. *P < 0.05 relative to the untreated Co set arbitrarily at 100. Note that in PAR2−/− mice none of the mean values from TGF-β1-treated cells were significantly different from Co.