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Fig. 6. PRRX1 promotes NRG1 expression in human EPDCs. (A) Schematic of the workflow for the experiments shown in B and C. After isolation, human fetal epicardial cells are cultured in the presence of the ALK4/5/7 kinase inhibitor SB-431542. Cells transform from cobble- to spindle-shape upon removal of SB-431542. (B) Representative brightfield pictures of cobble- and spindle-shaped human fetal epicardial cells. Scale bars: 100 µm. (C) qPCR results for POSTN, FN1, PRRX1 and NRG1 in human fetal cobble (n=3) and spindle (n=3) epicardial cells (mean±s.d.; POSTN P<0.001, FN1 P=0.001, PRRX1 P=0.01, NRG1 P<0.001, unpaired t-tests). (D) Schematic of the workflow for the experiments shown in E and F. (E) Western blot for PRRX1 in U87 cells. Vinculin was used as a loading control. (F) qPCR results for PRRX1 and NRG1 in human fetal spindle epicardial cells after PRRX1 siRNA treatment (non-transfected cells n=4, CTRL siRNA n=4, PRRX1 siRNA n=4) (mean±s.d., PRRX1 CTRL siRNA versus PRRX1 siRNA P=0.003, NRG1 CTRL siRNA versus PRRX1 siRNA P=0.04, unpaired t-tests) (G) ELISA results for secreted NRG1-β1 in the conditioned cell culture medium of human fetal spindle epicardial cells between 24 and 48 h after PRRX1 siRNA treatment (non-transfected cells n=3, CTRL siRNA n=3, PRRX1 siRNA n=3) (mean±s.d., CTRL siRNA versus PRRX1 siRNA P=0.0061, unpaired t-tests).