Fig 2: Visualization of phosphorylation of PDGFR-ß in BJ hTert cells. (a) Images comparing serum-starved BJ hTert cells (-) versus cells treated with PDGF-BB for 45 min on ice (+) by detecting phosphorylation levels of the PDGFR-ß (anti-PDGFR and anti-pan phosphorylation (pY100) antibodies). The RCA products were labeled with Cy3 (red dots in the merged images) and the nuclei were stained with Hoechst 33342. Scale bar (white) = 50 µm. (b) Quantification of signals comparing conventional and UnFold in situ PLA at different probe concentrations. The experiment was performed four times. Five images were collected per experimental condition. Error bars represent SEM.

Fig 3: PDGFR-a/ß signaling in sarcoma CSCs contributes to chemotherapy resistance in vitro.Proliferation assays for human sarcoma cell lines grown as (a) monolayers or (b) spheroids for 48–60 h. Treatment groups included cells with PDGFR-a knockdown (sh.PDGFR-a), PDGFR-ß knockdown (sh.PDGFR-ß), and cells treated with imatinib; each group was also treated with doxorubicin chemotherapy (Dox) 0.5 µmM or DMSO. Proliferation assays were also performed with both PDGFR-a and -ß double knockdown cells (sh.PDGFR-a/ß) in human sarcoma cells grown as (c) monolayers or (d) spheroids, following treatment with doxorubicin or DMSO. Experiments in a–d were performed three times with similar results. *p < 0.05 compared to control and monotherapy groups

Fig 4: HMGB1 secretion via PDGFR-ß signaling pathway in MS-stimulated VSMCs.(A) VSMCs were transfected with PDGFR-a or PDGFR-ß siRNA (200 nM) for 48 hrs, and then stimulated by 3% MS for 1 hr. The expression levels of phosphorylated and total PDGFR isoforms (PDGFR-a and PDGFR-ß) were determined by Western blotting using ß-actin as an internal control. Quantitative results were expressed as the mean ± SEM of 3–4 independent experiments. *P < 0.05 and **P < 0.01 vs. corresponding value in control, #P < 0.05 vs. corresponding value in negative control. (B) VSMCs were transfected with PDGFR-a or PDGFR-ß siRNA (200 nM) for 48 hrs, and then stimulated by 3% MS for 1 hr. HMGB1 secreted into the culture media was determined by ELISA, and results were expressed as the mean ± SEM of 6 independent experiments. **P < 0.01 vs. corresponding value in control, ## P < 0.01 vs. corresponding value in vehicle.

Fig 5: 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-a-SMA, and anti-ß-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-a-SMA, and anti-ß-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-a (p-PDGFR-a), anti-PDGFR-a, anti-phospho-PDGFR-ß (p-PDGFR-ß), anti-PDGFR-ß, 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-ß-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