Figure 8.

SMAD3 activation in untreated RNS GFs and treated control and RNS GFs. Immunocytochemical staining of control (A,D) and RNS (B,C,E,F) GFs cultured without TGFβ1 (A–C) or with 5 ng/ml TGFβ1 (D–F) for 6 h. Cells were fluorescently labeled for p-SMAD3 (green), nuclei (blue), and the specific F-actin marker, phalloidin (red). Co-localization of p-SMAD3 and nuclei indicated nuclear translocation of p-SMAD3 (green arrow). (A) In control untreated GFs, p-SMAD3 immunoreactivity is detected at low levels in some nuclei. (B,C) In RNS untreated GFs, p-SMAD3 was increased in intensity in all nuclei. (D–F) TGFβ1 induced an increase in p-SMAD3 nuclei in normal (D) and mutant GFs (E,F). (G) Western blot were performed on cell lysates. P-YAP (Ser 397) protein levels were decreased in RNS GFs without or with TGFβ1 compared to Control. TAZ protein levels were increased in RNS GFs compared to normal GFs. Densitometric analysis of Phospho-YAP and TAZ bands normalized to corresponding GAPDH bands. Data represent mean fold change in band intensity ± s.d. relative to GAPDH of 3 independent experiments in triplicates. Data were analyzed by one-way ANOVA with Bonferroni multiple comparisons test (**p < 0.01, ***p < 0.001). (H) Western blots were performed on cell lysates. Alpha-SMA protein levels were increased in controls GFs cultured with TGFβ1. Alpha-SMA protein levels were increased in RNS GFs cultured without or with TGFβ1 compared to control. Densitometric analysis of a-SMA bands normalized to corresponding GAPDH levels. Scale bars: (A-F) = 20 µm.