Fig 2: PDGFR activity is required for optimal CXCR4 signaling in SHH MB cells and GRK6 is downregulated and differentially expressed in MB. (A) Left panel: Daoy cells were starved and treated with or without 1 µg/ml anti-PDGFRß blocking antibody for 24 h, then stimulated by 10 ng/ml PDGF-BB or 100 ng/ml CXCL12 (SDF-1a) for 15 min. Western blot of P-ERK shows PDGFR function is required for optimal CXCR4 signaling; Right panel: Quantitative analysis of Western blot shows CXCL12-induced P-ERK is significantly decreased by PDGFRß blocking antibody (P < 0.05, lane 3, 1.53 ± 0.12 vs. lane 6, 1.09 ± 0.15, three independent experiments). (B) 29 human MB specimens were collected and used for microarray analysis. Average expression level of GRK6 mRNA is lowest among the detectable GRKs, (P < 0.01). (C) Microarray database of 9 human metastatic and 14 non-metastatic MB showed that the percentage of tumors with detectable GRK6 mRNA was notably decreased in metastatic MB (M+, 22%), compared to non-metastatic MB (M0, 43%). However, the percentage of tumors with detectable GRK5 mRNA was not appreciably different in M + (22%), vs. M0 (29%).

Fig 3: Inhibiting cell-autogenous PDGF signaling enhances adipogenesis. (a) Oil red O staining of hemSCs exposed to adipogenic media with or without PDGFR inhibitors, AG-370 and AG-1296, and PDGFR neutralizing antibodies. (b) Quantitative analysis of C/EBPa and PPAR? in cells exposed to PDGF inhibitors as in (a) (data presented relative to control media; *P<0.05 compared with control media; †P<0.05 compared with adipogenic media; n=3). (c) Effect of PDGFR-a and PDGFR-ß-neutralizing antibodies and chemical inhibitors on reversing the inhibition by exogenous PDGF-BB (*P<0.05 compared with PDGF-BB treatment)

Fig 4: Pericyte soma on WM capillaries demonstrated by immunofluorescence. A–F. PDGFR-ß (red) and COL4 (green) immunofluorescence staining with nuclei (DAPI) (blue). A,B. Low- and high-power images showing capillary segments (arrowheads) with overlapping PDGFR-ß (red), COL4 (green) and DAPI (blue). C. Same vessel segment as B with PDGFR-ß (red) and COL4 (green); D. COL4 (green) and DAPI (blue); E. PDGFR-ß (red) and DAPI (blue). F. Another capillary segment with PDGFR-ß (red), COL4 (green) and DAPI (blue) clearly showing pericyte cell body. G–J. Images taken by a confocal microscope showing pericyte cell bodies (arrows) with nuclear stain (DAPI). Capillaries and pericyte processes are revealed by COL4 and PDGFR-ß (red) immunoreactivities. Magnification bars: A = 50 µm, F, J = 10 µm.

Fig 5: GRK6 expression is negatively regulated by GF/PDGFR. (A) Quantitative real time RT-PCR was performed to determine the expression of GRK6 mRNA in medulloblastoma cells with normal vs. downregulated PDGFR expression. (A) GRK6 mRNA is increased 1.5 folds in B9 (PDGFRß down-regulated by shRNA in D556, shown below left panel) and 1.5 folds in A4 (PDGFRß down-regulated by shRNA in Daoy, shown below right panel) respectively, compared to the parental cells (D556 NC1 or Daoy NC1), P < 0.05 (N = 3). (B) Protein level of GRK6 is increased in D556 (B9) and Daoy (A4) with or without CXCL12 treatment. (C) D556 or Daoy was cultured in EMEM medium containing 10% serum (GF+) or serum-free EMEM (GF-) for 24 h, then added 10% serum to the cells cultured in serum-free medium (GF add-back) and cells grown for 24-72 h. Cell lysates were harvested for Western Blot. GF withdrawal results in robust increase of GRK6 in D556 and Daoy (left and middle panels). In right panel, Daoy cells were starved for 24 h and then treated with 10 ng/ml PDGF-BB for 24 h. GRK6 level determined by Western blot shows marked decrease with PDGF treatment.