Fig 2: The inhibitory effect of sunitinib on PDGF-induced intracellular signaling in vitro and in vivo. (A) Left panel: RSMCs were stimulated with 20 ng/ml PDGF-BB and 0–10 µM sunitinib in serum-free medium for 24 h. Western blot analysis of p-ERK, p-PDGFR and GAPDH, which served as a loading control, was performed. (B) Quantification of western blots demonstrated that protein expression levels of p-PDGFR were decreased by sunitinib treatment in a dose-dependent manner, and PDGF-induced downstream p-ERK protein expression levels were suppressed. *P<0.05 and **P<0.01 vs. vehicle. (C) The media, adventitia and neointima were harvested from rabbits implanted with BMS or STES, and western blot analysis of p-ERK was performed, with GAPDH serving as a loading control. (D) Quantification of western blots demonstrated that protein expression levels of p-ERK in the neointima were significantly decreased in STES compared with BMS. *P<0.05 vs. BMS Neo. PDGF, platelet-derived growth factor; RMSCs, rat smooth muscle cells; p-, phosphorylated; ERK, extracellular signal-regulated kinase; PDGFR, PDGF receptor; BMS, bare-metal stent; STES, sunitinib-eluting stent; Med, media; A, adventitia; Neo, neointima; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Fig 3: CD44s mediates CSC-like properties through the PDGFRβ/Stat3 signaling pathway. (A) Gene ontology analysis of CD44s-correlated genes. (B) STRING analysis showing functional association networks of CD44s-correlated genes. (C) GSEA plots showing the enrichment of the PDGF pathway signature in the CD44s-correlated gene list. (D) The levels of p-PDGFRβ, p-Stat3, and p-ERK were examined in HMLE-Twist control and shCD44 cells using immunoblot analysis. Cells were treated with 10 ng/mL PDGF for the indicated time intervals. The relative intensity of phosphorylated to unphosphorylated signals is depicted below the images. Representative images are shown from three biological replicates. (E) 293FT cells cotransfected with CD44s and Flag-tagged PDGFRβ were subjected to immunoprecipitation (IP). (F) The levels of p-PDGFRβ were examined using immunoblot analysis in HMLE/Twist cells expressing control and ESRP1 cDNA or coexpressing ESRP1 and CD44s cDNA. Cells were treated with 10 ng/mL PDGF for 15 min. The relative intensity of p-PDGFRβ to total PDGFRβ is depicted below the images. Representative images are shown from three biological replicates. (G) Mammosphere-forming ability assay in control and CD44s-expressing Mes10A cells that were treated with 5 µM PDGFR inhibitor IV or 10 µM Stat3 inhibitor XVIII. (H) Mes10A cells expressing control and CD44s cDNA were pretreated with 5 µM PDGFR inhibitor IV or 10 µM Stat3 inhibitor XVIII for 1 h followed by treatment with 100 µM cisplatin (Cispl) for 24 h. Cell death was assessed and plotted as percent dead cells. (I) Mes10A cells expressing CD44s cDNA were treated with 10 µM Stat3 inhibitor XVIII or 5 µM PDGFR inhibitor IV for 48 h. The expression level of E-cadherin and N-cadherin was examined using immunoblot analysis. The parental Mes10A cells were used as control. Representative images are shown from three biological replicates. Error bars in G and H indicate SEM. n = 3. (*) P < 0.05; (**) P < 0.01.

Fig 4: Glycosylation at Asn254 is required for the PDGF-C activation and signaling of PDGF-C. (A) Glycosylation at Asn254 is required for the PDGF-C activation. HEK293 A cells were transfected with plasmids carrying wild-type (WT) or mutant forms of PDGF-C. Two days later, CMs were collected, concentrated, and analyzed by Western blot with antibodies that detected full-length and the core-domain of PDGF-C. (B) Glycosylation at Asn254 was required for PDGF-C–mediated signaling via PDGFR-α and-β. CMs were collected from transfected HEK293 A cells as described in A and added to cultured NIH 3T3 cells for 5 min. NIH 3T3 cells were harvested and analyzed by Western blot.

Fig 5: Activation of PDGFR‐β in mesenchymal cells in Foxd1Cre::Pdgfrb +/J mice results in progressive interstitial fibrosis, inflammation, and tubular damage A, B(A) Histological stainings and its histomorphometric quantification (B) of kidney cortex for α‐SMA of wt (25 weeks) and Foxd1Cre::Pdgfrb +/J mice 6 and 35 weeks of age show expansion of myofibroblasts in the interstitium of the transgenic mice.C, D(C) Collagen I staining and its histomorphometric quantification (D) show the progression of interstitial fibrosis in the time course.E, FThe tubular stress marker lipocalin‐2 (LCN2) is not expressed in early time points but increases in the time course in late stages when fibrosis is more prominent, as seen on histological staining (E) and mRNA level (F).G, HA slight, but significant, increase in interstitial ErHr3‐positive stained macrophages was observed, particularly at later stages (G). For evaluation, ErHr3‐positive cells were counted on 20 view fields on 40× magnification (H).Data information: Scale bars = 50 µm. Bar graphs show means ± SD of n = 3 animals per group. Statistical analysis was performed by unpaired two‐tailed Student's t‐test. *P < 0.05 compared to wt of the same time point. Exact P‐values are provided in Appendix Table S4.Source data are available online for this figure.