NOX4-Derived ROS Promotes Collagen I Deposition in Bronchial Smooth Muscle Cells by Activating Noncanonical p38MAPK/Akt-Mediated TGF-β Signaling

Figure 3

Effects of TGF-β₁ on the expression of NOX4, α-SMA, and collagen type I in HBSMCs. (a, c, e) Cells were incubated with TGF-β₁ (2 ng/mL) for the indicated time. (b, d, f) Cells were stimulated with various concentrations of TGF-β₁ (0.5-10 ng/mL) for 24 h (mRNA levels) or 36 h (protein levels). (a, b) The mRNA levels of NOX4 were determined by real-time Q-PCR. (c, d) The mRNA levels of α-SMA were determined by real-time Q-PCR. (e–h) The protein expression of NOX4, α-SMA, and collagen I was determined by Western blot. (i) Double immunofluorescence staining for colocalization of NOX4 (green) and α-SMA (red) in HBSMCs. DAPI (blue) labeled the nuclei. (j) Representative images of immunofluorescence staining of α-SMA (green) and collagen I (green) expression. Nuclei were labeled with DAPI (blue) in cells treated with/without 2 ng/mL TGF-β₁ for 36 h. Data was represented as from three independent experiments and presented as . , , and compared to the unstimulated control. ^# compared to the unstimulated control. ^& compared to the unstimulated control.

(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

(i)

(j)