Fig 2: In situ injection of PDGFB or PDGFD induces fibrosis in uninjured spinal cord. A–L Immunofluorescence staining of PDGFB (red) and PDGFRß (green) in sagittal and coronal sections of the Control and PDGFB groups at 7 days after injection. M–X Immunofluorescence staining of PDGFD (red) and PDGFRß (green) in sagittal and coronal sections of the Control and PDGFD groups at 7 days after injection. Y–D’ Immunofluorescence staining of PDGFRß (green), fibronectin (red) and laminin (violet) in sagittal sections of the PDGFB and PDGFB + SU16f groups at 7 days after injection. E’–J’ Immunofluorescence staining of PDGFRß (green), fibronectin (red) and laminin (violet) in sagittal sections of the PDGFD and PDGFD + SU16f groups at 7 days after injection. Arrowheads indicate the sites of in situ injection. Scale bars: 200 µm

Fig 3: Spatiotemporal distribution of PDGFD and PDGFRß after SCI. Immunofluorescence staining of PDGFD (red), PDGFRß (green) and nuclei (blue) in sagittal sections before SCI and at 3, 7, 14 and 28 dpi. The region of interest (ROI) represents the boxed region on the left. Asterisks indicate the injured core. Scale bars: 200 µm in S and 20 µm in T

Fig 4: PDGFD is mainly secreted by macrophages/microglia and fibroblasts after SCI. A–D Immunofluorescence staining of PDGFD (red), CD68 (green) and PDGFRβ (white) in sagittal sections at 14 dpi. E–H Higher magnification images of the boxed region in D. Arrows indicate CD68+PDGFD+ cells. Arrowheads indicate PDGFRβ+PDGFD+ cells. I–L Immunofluorescence staining of PDGFD (red), CD31 (green) and nuclei (blue) in sagittal sections at 14 dpi. J–L represents boxed region in I. M–P Immunofluorescence staining of PDGFD (red), GFAP (green) and nuclei (blue) in sagittal sections at 14 dpi. N–P represents boxed region in M. Q Quantification of the proportion of CD68+PDGFD+ cells, PDGFRβ+PDGFD+ cells or CD31+PDGFD+ cells in PDGFD+ cells at 14 dpi. Asterisks indicate the injured core. Scale bars: 200 μm in D, I and M and 10 μm in H, L and P. n = 5 animals per group

Fig 5: HIF1a regulates the expression of PDGFRA and PDGFD in GBM cells. A WT U251 cells were cultured in the indicated hypoxic conditions and protein expression was detected by Western blot. B Co-expression of HIF1a with PDGFRa and PDGF-D in GBM tissue arrays were examined by immunofluorescence co-staining. C-F. HIF1a directly regulates PDGFRA promoter activity. C Diagram of 5' region of the PDGFRA gene, with positions of three HRE sites, its proximal promoter region, intronic enhancer regions and locations of ChIP primers, is shown. D Induction of PDGFRA promoter activity by HIF1a. HEK293 cells were co-transfected with plasmids of PDGFRA promoter and either empty vector control or HIF1a-PPN. Twenty-four hours after transfection, GFP expression was visualized by fluorescence microscopy, depicted in the representative photographs. PDGFRA-P1 corresponds to the promoter construct containing promoter alone; E1, E2 denote either of the intronic enhancers. E GFP mean fluorescence intensity (MFI) was quantitated by flow cytometry and summarized for cells transfected in triplicate. F HIF1a binds to the enhancers of PDGFRA promoter. ChIP assay was conducted in U251 cells using rabbit HIF1a antibody and rabbit IgG as a parallel control. One tenth of lysate was used as the input and the data shown is representative of 3 experiments. G, H Induction of PDGFD promoter activity by HIF1a was performed as in D,E. I HIF1a binds to the proximal HRE site of the PDGFD promoter as in F. The SD is from three independent experiments each performed in triplicate