Figure 9. Berbamine induces FoxA2 to regulate TGFβ signaling and control EC-like myofibroblast differentiation.

a, Expression of ALK5 in tdTomato+VE-cadherin+ cells isolated from the lungs of SM22a^creRosa^tdTomato mice and treated with berbamine (20 μM).

b, Gene expression in tdTomato+VE-cadherin+ cells transfected with MGP siRNA in combination with berbamine treatment with or without overexpression of ALK5 (n=5). CMV, cytomegalovirus promoter. Fn1, fibronectin 1. SCR, scrambled siRNA.

c, Expression of FoxA2 in tdTomato+VE-cadherin+ cells isolated from the lungs of SM22a^creRosa^tdTomato mice and treated with berbamine (20 μM).

d, Immunoblotting with densitometry using tdTomato+VE-cadherin+ cell lysates treated with berbamine (Bbm) with or without overexpression of FoxA2 (CMV-FoxA2) (n=3).

e, Expression of endothelial markers in tdTomato+VE-cadherin+ cells treated with berbamine in combination with FoxA2 overexpression or FoxA2 knockdown (FoxA2 siRNA) (n=6).

f, Immunoblotting with densitometry of the lungs of VE-cadherin^creMgp^flox/flox mice, Sm22a^creMgp^flox/flox mice and control mice after treatment of berbamine (Bbm) (n=6).

g, Quantification of VE-cadherin positive and PDGFRα positive (VE-cadherin+PDGFRα+) cells and VE-cadherin negative and PDGFRα positive (VE-cadherin-PDGFRα+) cells after immunostaining of the lungs of bleomycin-injected wild-type mice, where gene expression was examined by real-time PCR (n=8).

h, Immunostaining of ALK5 and FoxA2 of human pulmonary fibrosis (n=3).

a and c were analyzed for statistical significance by unpaired 2-tailed Student’s t test. b, e and g were analyzed for statistical significance by ANOVA with post hoc Tukey’s test. The bounds of the boxes are upper and lower quartiles with data points. The line in the box is median. Error bars are maximal and minimal values. ***, P<0.0001.