Table 1.
Human Primer Sequences Used for Realtime RT-PCR.
Figure 1.
Inhibitory effect of pirfenidone on proliferation of SRA01/04 cells as measured by MTT assay.
Cells with 10% FBS were treated with 0, 0.01, 0.1, 0.2, 0.3, 0.5, or 1.0 mg/mL pirfenidone for 0, 4, 12, 24, 48, or 72 hours. Data are derived from the mean ± SD of triplicate results in three independent experiments. *P<0.05, and **P<0.001 from comparisons between cells with treatment and control cells with 10% FBS, at different time points.
Figure 2.
Cell viability of SRA01/04 cells after treated with pirfenidone for 24 hours in a trypan blue exclusion test.
After 24 hours’ treatment with PFD (0, 0.3, 0.5, and 1 mg/ml), the percentages of living cells were more than 90% in all groups. No statistically significant difference was found between PFD treated and control groups (P>0.05). Data are the mean ± SD of triplicates from an experiment that was repeated with similar results.
Figure 3.
Cytotoxicity of pirfenidone in a LDH assay.
After 24 or 48 hours’ treatment with PFD (0, 0.25, and 0.5 mg/ml), no statistically significant difference was found between the percentage of cell-mediated lysis in PFD treated groups and control group (P>0.05). Data are the mean ± SD of triplicates from an experiment that was repeated with similar results.
Figure 4.
Inhibitory effect of pirfenidone on TGF-β2-induced fibroblastic phenotypes in SRA01/04 cells.
There was no apparent morphological change in SRA01/04 cells after 24 hours’ treatment with 0.5 mg/ml PFD only (B) compared with control cells(A). In the presence of 12.5 ng/ml TGF-β2, the TGF-β2-induced morphological changes in SRA01/04 cells (C) were detected, including elongated and spindle-like shapes, which were noticeably suppressed by co-treatment with 0.5 mg/ml PFD (D).
Figure 5.
Inhibitory effect of pirfenidone on TGF-β2-induced epithlial-mesenchymal transition in SRA01/04 cells.
Compared with control cells (A), immunocytofluorescence demonstrated that the TGF-beta2 significantly up-regulated the mesenchymal phenotypic marker fibronectin (FN, green) in SRA01/04 cells (C). Either in the absence (B) or presence (D) of TGF-beta2, FN was significantly down-regulated after 24 hours’ treatment with 0.5 mg/ml PFD. The nuclei stained by DAPI (blue). Magnification, ×400.
Figure 6.
Inhibitory effect of pirfenidone on TGF-β2-induced migration of SRA01/04 cells.
Light microscope images show decreased migratory ability of cells at 24 hours, after scratches were applied to the cells with 0, 0.25, or 0.5 mg/mL pirfenidone in the absence or presence of TGF-β2. *P<0.05 and **P<0.01 versus the corresponding value for control cells. Data in each bar are the mean ± SD of cells that migrated through the membrane in three separate experiments. Magnification, ×40.
Figure 7.
Inhibitory effect of pirfenidone on expression of TGFβ2 and SMADs mRNA in SRA01/04 cells.
Real-time RT-PCR showed that pirfenidone reduced the levels of TGFβ2, SMAD3, and SMAD4 when compared with control cells. **P<0.01 versus the corresponding value for control cells. Data are the mean ± SD of triplicates from an experiment that was repeated with similar results.
Figure 8.
Inhibitory effect of pirfenidone on expression of TGFβ2 and SMADs protein in SRA01/04 cells.
Western blot showed that pirfenidone reduced the protein levels of TGFβ2, SMAD3 and SMAD4 when compared with control cells. *P<0.05 and **P<0.01 versus the corresponding value for control cells. Data are mean ± SD of results from three independent cultures.
Figure 9.
Expression of TGFβ2 and SMADs protein in SRA01/04 cells after treated with pirfenidone by immunocytochemical detection under confocal microscopy.
TGFβ2 (A), SMAD3 (B), and SMAD4 (C) in the cytoplasm (red) and nuclei (blue) of HLECs were stained by immunocytochemistry after treatment with 0.25 (A2, B2, C2) and 0.5 mg/mL (A3, B3, C3) pirfenidone, respectively. (A0, B0, C0) Corresponding negative controls. Magnification, ×400.
Figure 10.
Inhibitory Effect of PFD on TGF-β2-induced Expression of SMADs protein.
Western blot showed that pirfenidone (0.5 mg/ml) reduced the TGF-β2-induced Expression of SMAD2 and SMAD3 when compared with control cells. The inhibitory effects could be detected but were not statistically significant in the 0.25 mg/ml PFD groups. *P<0.05 versus the corresponding value for control cells. Data are mean ± SD of results from three independent cultures.
Figure 11.
Inhibitory effect of pirfenidone on TGF-β2-induced nuclear translocation of SMADs in SRA01/04 cells under confocal microscopy.
After stimulated by 12.5 ng/ml TGF-β2 for 24 hours, the staining of nuclei SMAD4 (green) enhanced when that of SMAD4 in the cytoplasm were weakened, and SMAD2/3 proteins (red) were assembled on the nuclear membrane and some of them entered the nuclei. Expression of nuclei SMAD2/3 and SMAD4 were depressed after treatment with 0.5 mg/ml pirfenidone. The nuclei stained by DAPI (blue). Magnification, ×1000.