Fig 2: KIF3A knockdown increases α-SMA-positive myofibroblasts among SiO2-activated MRC-5 fibroblasts. (A) Western blot showing the effects of NC-siRNA and KIF3A-siRNA on expression of KIF3A, Ac-α-Tub, and ARL13B proteins in MRC-5 fibroblasts. GAPDH was used as a loading control (n=3). (B) Densitometric analyses of KIF3A, Ac-α-Tub, and ARL13B protein expression in MRC-5 fibroblasts. *P<0.05; **P<0.01. Data are the mean±SD. Statistical analysis was performed using one-way ANOVA and SPSS 20.0. (C) IF assay showing primary cilia in MRC-5 fibroblasts treated with NC-siRNA or KIF3A-siRNA. Primary cilia were labelled with an anti-Ac-α-Tub antibody. Scale bar=25 and 5 μm. (D) Treatment regimen of KIF3A knockdown in MRC-5 fibroblasts. MRC-5 fibroblasts were stimulated with SiO2 or serum-free medium (n=3 per group) for 12 h, and then transfected with NC-siRNA or KIF3A-siRNA until 36 h. (E) Expression of α-SMA in MRC-5 fibroblasts measured by IF. Scale bar=100 μm. (F, G) Western blot and densitometric analyses of the effects of NC-siRNA and KIF3A-siRNA on expression of COL I, α-SMA, MRTF-A and SRF proteins in MRC-5 fibroblasts with or without SiO2 stimulation. α-Tub was used as a loading control (n=3). *P<0.05; **P<0.01. Data are the mean±SD. Statistical analysis was performed using one-way ANOVA and SPSS 20.0.

Fig 3: Observation of primary cilia in MRC-5 fibroblasts treated with SiO2. (A) IF showing changes in primary cilia of MRC-5 fibroblasts treated with SiO2. Primary cilia were labelled with an anti-Ac-α-Tub antibody. Scale bar=5 μm. Quantification of the frequency (B) and length (C) of primary cilia under the indicated conditions (mean±SD, n=3, >100 cells/experiment). (D) Western blot showing the effects of SiO2 (50 μg/cm2) on expression of ARL13B and Ac-α-Tub in MRC-5 fibroblasts. GAPDH was used as a loading control (n=3). (E) Densitometric analyses indicating that SiO2 induced ARL13B and Ac-α-Tub expression in MRC-5 fibroblasts. *P<0.05 vs. SiO2 stimulation at 0 h; #P<0.05 vs. SiO2 stimulation at 12 h. Data are the mean±SD. Statistical analysis was performed using one-way ANOVA and SPSS 20.0.