Figure 5.

Knock-down of OGR1 expression does not have additive or synergistic effects on TGF-β induced myofibroblast differentiation. Human fibroblasts (healthy: A–D, IPF: E–H) were transfected with either non-targeting (con) or OGR1 siRNA and were subsequently treated with either TGF-β1 or DMSO as a control. Protein lysates were then harvested and western blotting was performed with antibodies against OGR1, α-SMA, collagen 1A1, and beta-tubulin (as a loading control). Representative western blots are presented (A and E, respectively), and densitometry analysis is graphed below. OGR1 was successfully knocked down in both healthy (B, ** p = 0.0010 and 0.0017) and IPF-derived fibroblasts (F, *** p = 0.0007 and 0.0009). As previously demonstrated, treatment with TGF-β1 decreased basal OGR1 expression in IPF-derived fibroblasts (** p = 0.0071). As expected, TGF-β1 significantly increased expression of α-SMA in both healthy (C, *** p = 0.0003 and ** p = 0.0028) and IPF-derived fibroblasts (G, ** p = 0.0076 and * p = 0.02). Interestingly, in healthy fibroblasts, decreasing basal expression of OGR1 caused a significant increase in α-SMA expression (C, * p = 0.0373). Again, as expected, treatment with TGF-β1 caused increased collagen1A1 expression in healthy (D, * p = 0.0364) and IPF fibroblasts (H, *** p = 0.0037 and ** p = 0.0001, respectively). In IPF fibroblasts, decreasing basal expression of OGR1 caused increased collagen1A1 expression (H, *** p = 0.0003). Suppressing OGR1 expression did not add to the ability of TGF-β to induced myofibroblast differentiation. Data represent mean expression ± SEM.