Cd133

The following primary antibodies were used in this study: rabbit anti-mouse Notch4, rabbit anti-mouse Hey1, rabbit anti-mouse RBPJ, rabbit anti-mouse Notch1, rabbit anti-mouse Notch2, rabbit anti- mouse Notch1, rabbit anti- mouse CK7, rabbit anti- mouse CK19, rabbit anti- mouse AFP, mouse anti-goat Hes1, rabbit anti-mouse Notch3 (Sigma), CD133(Abcam).

Immunofluorescence staining was performed as previously described (23 (link)). First, the GBM cells were fixed with 4% paraformaldehyde (Solarbio, Beijing, China) for 10 min, permeabilized with 0.5% Triton X-100 (Solarbio) for 20 min, blocked with 5% bovine serum albumin (Solarbio) for 1 h, and probed with primary antibodies to CD133, nestin, GFAP, βIII-tubulin, E-cadherin, and vimentin (1:100; Abcam) at 4°C overnight. Then, all GBM cell samples were treated with fluorescein isothiocyanate or rhodamine-conjugated secondary antibodies. Subsequently, the cells were counterstained with 4ʹ,6-diamidino-2-phenylindole (Sigma-Aldrich, Shanghai, China). Finally, the staining was visualized using a laser scanning confocal microscope (Olympus).

Cells were lysed with M-Per Mammalian Protein Extraction Reagent (Pierce, Rockford, IL). Equal amounts of the extracts were loaded, subjected to 10% SDS-PAGE, transferred onto nitrocellulose membranes, and probed by antibodies against STAT3 (Cell Signaling), E-cadherin (Cell Signaling), CD133 (Abcam), MEF2D (Abcam), ROCK1 (Abcam), WNT7A (Abcam), KPNA4 (Abcam), VEGF-A (Santa Cruz), Snail (Santa Cruz), Survivin (Santa Cruz) and GAPDH (Santa Cruz) at 4 °C overnight. After incubation with the corresponding secondary antibodies, signals were detected with an ECL detection kit (Amersham Pharmacia Biotech, UK).

After the cells were made into a single-cell suspension with TrypLE (Gibco, USA), loading buffer containing 1% β-mercaptoethanol (Solarbio, China) was added to lyse the extracted proteins. Proteins were subsequently loaded into 10% SDS-PAGE and transferred to PVDF membranes. We blocked with TBST containing 3% bovine serum albumin for one hour, followed by incubation with primary antibody overnight at 4°C. Primary antibodies are as follows: Oct4 (Zen-BioScience, China), Nanog (Zen-BioScience, China), CD44 (Abcam, UK), CD133 (Abcam, UK), Ascl2 (CST, USA), Cleaved-PARP (CST, USA), Cleaved-Caspase3 (CST, USA), p21 (Abcam, UK), and β-actin (Beyotime, China). Membranes were rinsed, followed by secondary antibody (Beyotime, China) incubation for one hour, and bands were visualized using the ECL detection kit.

Single-cell suspensions (1 × 10⁶) of PBMC were analyzed for expression of lineage-negative (Lin−) CD34+ and CD133+ stem cell markers as we described^{24 (link),25 (link)}. All antibodies used were from commercial sources: CD133 (Abcam, Cambridge, U.K.); CD34 (R&D Systems, Minneapolis, MN); Cy3 (rabbit), FITC (goat) (eBioscience). Nonspecific antibody binding was blocked with donkey and mouse serum (Sigma-Aldrich) for 30 min. Cells were incubated with antibodies for 1 h at 4 °C, and the positive cells were counted by flow cytometry (fluorescence-activated cell sorting [FACS]) using CELLQuest software (Becton Dickinson, Franklin Lakes, NJ).

Whole-cell lysates were homogenized in modified RIPA buffer^{14 (link)} and assayed by immunoblot as previously described^{14 (link)}. The primary antibodies against FKBP51 (rabbit polyclonal; Novus Biological), FKBP51s^{13 (link)} and CD274/PD-L1 (rabbit polyclonal; Novus Biological) were used diluted 1:2500. CD133 (rabbit polyclonal; Abcam; Cambridge, UK) was used diluted 1:1000. A further antibody Pdcd-1L1 (rabbit polyclonal, Santa Cruz Biotechnology; Santa Cruz, CA, USA) was used for PD-L1 detection at the 1:1000 dilution. Antibody against M2-Flag, caspase-7 and γ-Tubulin (mouse monoclonal; Sigma-Aldrich, St. Louis, MO, USA) were used diluted 1:5000. Anti β-Actin-Peroxidase (mouse monoclonal; Sigma-Adrich) was used diluted 1:10000. Anti-phospho-Akt (Ser473), Akt1/2/3, phosphor-S6 kinase, G3PDH and Sox-2 (rabbit monoclonal; Cell Signaling, Danvers, MA, USA) were used diluted 1:1000. Anti-p70S6 kinase (rabbit polyclonal; Santa Cruz) was used diluted 1:500.

To extract total protein, sodium dodecyl sulfate (SDS) lysis buffer (1% SDS, 60 mM Tris-HCl) with protease inhibitor (Roche, Basel, Switzerland) and phosphatase inhibitor (GenDEPOT, Katy, TX, USA) was used. WB was performed as previously reported [28 (link)]. The primary antibodies used were as follows: hexokinase 2 (HK2, ab209847, EPR20839, 1:500), glucose transporter 1(GLUT1, ab652, 1:2000), L-type amino acid transporter 1 (LAT1, ab208776, EPR17573, 1:1000), vascular endothelial growth factor B (VEGFB, ab110649, EPR4555, 1:1000), VEGFC (ab135506, 1:2000), CD133 (ab19898, 1:1000) and Snail/Slug (ab180714, 1:1000) from Abcam (Cambridge, UK), signal transducer and activator of transcription 3 (STAT3, #9139, 1:1000), pSTAT3 (#9145, D3A7, 1:2000) from Cell Signaling Technology (Danvers, MA, USA), and β-actin-HRP (sc-47778, C4, 1:5000) from Santa Cruz Biotechnology (Dalla, TX, USA). For the digital visualization of the chemiluminescent WB, the ChemiDoc XRS (Bio-Rad, Hercules, CA, USA) was used. These experiments were replicated at least three times with similar results. ImageJ (National Institutes of Health, Bethesda, MD, USA) and BioRad Image Lab 6 (Bio-Rad) were used for band quantification.