Pdgfrα

The cells were lysed using RIPA lysis buffer (Beyotime, P0013B) with freshly added 1% phenylmethylsulfonyl fluoride (PMSF, Amresco, O754) solution. Protein concentration was determined using Coomassie brilliant Blue G-250. SDS-PAGE and Western blotting were carried out as reported previously (Niu et al., 2012a (link)). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes and visualized by chemiluminescence (ECL Plus, GE Healthcare, Marlborough, MA, USA) after incubation with the antibodies. β-actin was used as the loading control. Quantification of band intensity was performed using ImageJ software. The primary antibodies included the following: rabbit polyclonal anti-RyR3 (1:1000, Millipore), rabbit polyclonal anti-platelet-derived growth factor receptor α (PDGFRα; 1:1000, Santa Cruz, sc-338), mouse anti-2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase; 1:1000, Abcam, ab6319), goat anti-MBP (1:1000, Santa Cruz) and mouse anti-β-actin (1:2000, Santa Cruz, sc-47778). The secondary antibodies included the following: goat anti-mouse-HRP (1:2000, Santa Cruz, sc-2094), goat anti-rabbit-HRP (1:2000, Santa Cruz, sc-2313) and rabbit anti-goat-HRP (1:2000, Santa Cruz, sc-2020).

Primary antibodies against Snail, Slug, Vimentin, E-cadherin, PDGFA, PDGFB, PDGFRα, P-AKT, AKT, P-ERK, ERK, CXCR4 and β-actin were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-FOXO1 antibodies were purchased from Abcam (Cambridge, MA, USA). All secondary antibodies were obtained from Pierce (Rockford, IL, USA). Lipofectamine 2000 was obtained from Invitrogen. SRB was purchased from Sigma (St. Louis, MO, USA).

The tissues were fixed in 10% formalin, followed by dehydration in ethanol gradients, permeabilized with xylene and paraffin embedded. Sections with 5μm thickness were deparaffinized with xylene, hydrated with ethanol gradient and stained with hematoxylin and eosin (H&E) (MXB Biotechnologies). Immunohistochemical staining for Nestin (1:500; Millipore), Sox2 (1:500; Cell Signaling), Olig2 (1:500; Millipore), PDGFRα (1:100; Santa Cruz), GFAP (1:600; Sigma) and H3K27me3 (1:1000; Millipore) was performed. Endogenous peroxidase inactivation was carried out with 3% H₂O₂, and antigen retrieval was performed using heated sodium citrate treatment. Afterward, primary antibodies were added to each slide at appropriate dilutions, and the sections incubated with polyperoxidase-labeled secondary antibodies for 25 minutes. The final signals were developed using the 3, 3’-diaminobenzidine substrate (DAB) (MXB Biotechnologies). The sections were analyzed by optical microscopy after counterstaining with hematoxylin.

Coverslips, coated with PLO/Laminin (3ug/ml), were placed into 24-well plates and seeded with 4 × 10⁴ DIPG cells. After 48 hours, cells were fixed with 4% paraformaldehyde and blocked with 5% BSA, then incubated with primary antibody overnight at 4°C. After that, cells were rinsed with PBS and incubated with fluorescence-labeled secondary antibody (1:2000; Invitrogen) for 45min at room temperature. Meanwhile, cells were counterstained with DAPI. After rinsing with PBS, coverslips were mounted on slides with Fluoromount-G (SouthernBiotech). Images were obtained using a Zeiss microscope. Primary antibodies Ki67 (1:400; Thermo Scientific), Nestin (1:500; Millipore), GFAP (1:600; Sigma), Olig2 (1:500; Millipore) and PDGFRα (1:100; Santa Cruz) were used.

Brain regions of interest from mice of indicated ages were dissected on ice and homogenized in RIPA buffer (Pierce Biotechnology, MA) with protease and phosphatase inhibitor cocktails (Roche, QC). Homogenate was centrifuged at 9391 × g for 10 min at 4 °C. In all, 10–20 μg of the protein extract was separated by SDS–PAGE, and transferred to polyvinylidene fluoride membranes. After blocking, blots were incubated with primary and corresponding secondary antibodies, and visualized with an enhanced chemiluminescence detection system. Bands were imaged and quantified using a ChemiDOC MP gel imaging system (Bio-Rad, CA). The following primary antibodies were used: PDGFA, Santa Cruz, 1:100; PDGFRα, Santa Cruz, sc-338, 1:200; MBP, Millipore, MAB386, 1:100 (neonatal tissue) or 1:500 (adult tissue); PLP, Santa Cruz, sc-98781, 1:400; actin, Cell Signaling, 4967, 1:10,000. The relative expression levels of a protein were quantified by normalization using actin levels. When quantifying western blot results, full size images without any saturation were used. In addition, images of longer exposure time were taken to ensure that the location of even the weakest band was clear when drawing the region of interest for analysis.