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

Figure 6. Ectopic expression of Smad binding competent ALK5 mutants can rescue TGF-β1-induced Smad activation, transcriptional activity, and cell migration.

Figure 6

A. Panc-1 cells were transiently transfected twice (on days 1 and 2) with 50 nM of either control siRNA (Co) or PAR2 siRNA and on day 2 additionally with either empty vector (vector), wild-type ALK5 (ALK5), kinase-active ALK5-T204D (ALK5-TD), or kinase-active ALK5 unable to bind Smads, RImL45-T204D (RImL45-TD). Forty-eight h after the second round of transfection, cells were stimulated with TGF-β1 for 1 h, lysed and analysed by immunoblotting for the indicated proteins. A densitometric analysis of underexposed replicas is shown below the immunoblots. Successful knockdown of PAR2, ALK5 and PAR1 expression was verified by qPCR (not shown). Note that RImL45-TD transfection in PAR2 depleted cells failed to increase p-Smad3C levels following TGF-β1 stimulation when compared to the respective vector control despite dramatic overexpression of RImL45-TD. B. As in A., except that cells received, in addition, p6SBE-Luc and pRL-TK-Luc and were stimulated with TGF-β1 for 24 h before they were subjected to dual luciferase assay. Data represent the normalised mean ± SD of six wells. The assay shown is representative of four experiments. C. Real-time cell migration assay of Panc-1 cells transiently transfected with control siRNA (siCo) or PAR2 siRNA (siPAR2), in combination with either empty vector or wild-type ALK5 (left-hand graph), or empty vector, ALK5-TD or RImL45-TD (right-hand graph). For the sake of clarity, only the data from TGF-β1-treated cells are shown. Significant differences between the siPAR2 + vector group (blue curves/B in both graphs) and the siPAR2 + ALK5 (red curve/D, left-hand graph) or the siPAR2 + ALK5-TD group (red curve/D, right-hand graph) were seen at 5:00 and 2:45, respectively, and all later time points. One representative assay out of three assays performed in total is shown. Letters to the right-hand side of the graph allow for a colour-independent identification of the various curves.