Fig. 2

IFN-γ induced epithelial-to-mesenchymal transition (EMT) in human nasal epithelial cells (hNECs). a hNECs in submerged conditions were cultured with each type of cytokines (100 ng/ml) and incubated for 48 h. EMT markers were traced. b Each data are expressed as normalized band intensity adjusted to β-tubulin, which serves as loading control. c Representative images of hNECs treated with indicative cytokines (100 ng/ml) for 48 h were stained with phalloidin, which labels F-actin. d Representative z-stack confocal photographs of differentiation markers were acquired. ALI-cultured cells were incubated with anti-ac-tubulin (for ciliated cells), anti-Mucin 5AC (for goblet cells) and anti-E-Cad antibodies, respectively. e hNECs in ALI conditions were cultured with indicated cytokines (100 ng/ml) and incubated for 48 h. EMT markers were traced. f Each data are expressed as normalized band intensity adjusted to β-tubulin, which serves as loading control. g Representative phase contrast images of hNECs were acquired after treatment of IFN-γ (100 ng/ml) in submerged or ALI conditions. h hNECs were treated with or without of IFN-γ (100 ng/ml) for 48 h in submerged conditions. Cells were immunostained against EMT markers (E-Cad, α-SMA) and visualized. In all immunoblot analysis, protein intensities (a, b, e, f) were quantified by ImageJ software (n = 1, three independent experiments). Representative western blots (a, e) are depicted and expressed as relative band intensity adjusted to the highest band intensity of each protein. Statistical significance (b, f) for normalized band intensity was determined by Student’s t-test. *P < 0.05; **P < 0.01. Data are shown as means ± s.e.m. Scale bars, 50 µm (c, d, g, h)