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Fig. 2 | Respiratory Research

Fig. 2

From: Pirfenidone inhibits myofibroblast differentiation and lung fibrosis development during insufficient mitophagy

Fig. 2

PARK2-mediated mitophagy activation by PFD in LF. a WB using anti-PARK2, anti-PINK1, and anti-β-actin of cell lysates from control (lane 1) and indicated concentrations of PFD (lane2 ~ 6) treated LF. Protein samples were collected after 24 h treatment with PFD. In the lower panels are the average (±SEM) taken from six independent experiments shown as relative expression. *p < 0.05. b LF were treated with PFD (500 μg/ml) and mRNA samples were collected after 24 h treatment with PFD (n = 4). Real-Time-PCR was performed using primers to PARK2 or β-actin, as a control. PARK2 mRNA expression was normalized to β-actin. Shown is the fold increase (±SEM) relative to control treated cells. *p < 0.05. c WB using anti-PARK2 and anti-TOM20 of cell lysates from PFD (500 μg/ml)-treated LF. Protein samples for mitochondrial fractions were collected after 24 h treatment. The lower panel is the average (±SEM) taken from three independent experiments shown as relative expression. *p < 0.05. d Colocalization analysis of confocal laser scanning microscopic images of TOM20 staining and EGFP-LC3. LF were transfected with pEGFP-LC3 with a concomitant non-silencing control siRNA or PARK2 siRNA. PFD (500 μg/ml) treatment was started 48 h post-transfection. Baf A1 (20 nM) treatment was started 6 h before fixation and LF were fixed after 24 h treatment with PFD. The images are high magnification (400X). (Bar = 20 μm). e WB using anti-LC3 and anti-β-actin of cell lysates from control siRNA (lane 1 ~ 4), PARK2 siRNA (lane 5 ~ 8) transfected LF. PFD (500 μg/ml) treatment was started 48 h post transfection and protein samples were collected after 24 h treatment. Protease inhibitor (E64d 10 μg/ml, pepstatin A 10 μg/ml) treatment was started 6 h before collecting cell lysates. In the right panel is the average (±SEM) taken from four independent experiments shown as relative expression. *p < 0.05. f WB using anti-PARK2 and anti-β-actin of cell lysates from PFD (500 μg/ml)-treated LF isolated from IPF lungs. Protein samples were collected after 24 h treatment. The lower panel is the average (±SEM) taken from six independent experiments shown as relative expression. *p < 0.05. g WB using anti-LC3 and anti-β-actin of cell lysates from control (lane 1, 2), PFD (500 μg/ml) (lane 3, 4) treated LF isolated from IPF lungs. LF were treated with PFD for 24 h and protease inhibitor (E64d 10 μg/ml, pepstatin A 10 μg/ml) treatment was started 6 h before collecting cell lysates. In the lower panel is the average (±SEM) taken from six independent experiments shown as relative expression. *p < 0.05. (H) Colocalization analysis of confocal laser scanning microscopic images of TOM20 staining and EGFP-LC3. LF isolated from IPF lungs were transfected with pEGFP-LC3 with a concomitant non-silencing control siRNA or PARK2 siRNA. PFD (500 μg/ml) treatment was started 48 h post-transfection. Baf A1 (20 nM) treatment was started 6 h before fixation and LF were fixed after 24 h treatment with PFD. The images are high magnification (400X). (Bar = 20 μm)

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