Fig. 4

PFD attenuates myofibroblast differentiation during insufficient mitophagy via inhibiting PDGFR/PI3K/AKT signaling pathway in LF. a Fluorescence intensity of CM-H2DCFDA staining for intracellular ROS production. PFD (500 μg/ml) treatment was started 48 h post-siRNA transfection and incubation with CM-H2DCFDA (10 μM) was started after 24 h treatment in LF. The fluorescence level in the control siRNA transfected cells without PFD was designated as 1.0. The panel is the average (±SEM) taken from five independent experiments shown as relative expression.* p < 0.05. b Photographs of Hoechst 33258 and MitoSOX Red fluorescence staining in LF. LF were transfected with control siRNA and PARK2 siRNA. PFD (500 μg/ml) treatment was started 48 h post-siRNA transfection and staining was performed 48 h transfection. (Original magnification, 200X) (c) WB using anti-type I collagen, anti-α-SMA, and anti-β-actin of cell lysates from control siRNA (lane 1, 2), PARK2 siRNA (lane 3, 4) transfected LF. NAC (1 mM) treatment was started 48 h post transfection and protein samples were collected after 24 h treatment. In the right panels are the average (±SEM) taken from six independent experiments shown as relative expression. *p < 0.05. d WB using anti-type I collagen, anti-α-SMA, and anti-β-actin of cell lysates from control siRNA (lane 1, 2), PARK2 siRNA (lane 3, 4) transfected LF. MitoTempo (100 μM) treatment was started 48 h post transfection and protein samples were collected after 24 h treatment. In the right panels are the average (±SEM) taken from six independent experiments shown as relative expression. *p < 0.05. e WB using anti-phospho-PDGFR-α (p-PDGFR-α), anti-PDGFR-α, anti-phospho-PDGFR-β (p-PDGFR-β), anti-PDGFR-β, anti-phospho-PI3K p85 (p-PI3K p85), anti-PI3K p85, anti- phospho-AKT (p-AKT), anti-AKT, anti-phopho-S6K (p-S6K), anti-S6K, anti-phospho-4EBP-1 (p-4EBP-1), anti-4EBP-1, and anti-β-actin of cell lysates from control siRNA (lane 1, 2) and PARK2 siRNA(lane 3, 4) transfected LF. PDF (500 μg/ml) treatment was started 48 h post transfection and protein samples were collected after 24 h treatment. In the lower right panels are the average (±SEM) taken from five independent experiments shown as relative expressions. *p < 0.05