TGF-β signaling regulates the expression of LEC-related genes in HDLECs. (A) Expression levels of Prox1 determined by immunostaining in HDLECs. HDLECs were untreated (left) or treated with TGF-β1 (1 ng/mL; middle) or TβR-I inhibitor LY364947 (3 μM; right) for 24 hours and subjected to immunocytochemical examination. Bars represent 50 μm. (B) Expression levels of Prox1 in HDLECs treated as described in panel A were determined by immunoblotting. α-Tubulin levels were monitored as a loading control for whole-cell extracts. (C-E) Expression levels of Prox1, LYVE-1, and PAI-1 mRNAs were analyzed by real-time PCR at 24 hours after TGF-β1 or TβR-I inhibitor treatment. In the right 3 columns, cells were treated with 1 μM cycloheximide (CHX) for 24 hours before they were treated with TGF-β1 or TβR-I inhibitor for 24 hours. Values were normalized to amounts of GAPDH mRNA. Error bars represent SD (*P < .05, **P < .01, ***P < .001).