Ab19898

Formalin-fixed xenograft specimens were embedded into paraffin. Sections (4 μm thickness) were prepared using a RM2145 rotary microtome (Leica Microsystems). For immunohistochemistry analysis, sections were incubated with anti-CD44 (ab157107, Abcam) or anti-CD133 (ab19898, Abcam) primary antibodies overnight at 4 °C. The sections were then treated with HRP-conjugated secondary antibody; diaminobenzidine (ScyTek) was used as a chromogen, and sections were lightly counterstained with hematoxylin (ScyTek).

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.

Colorectal cancer stem cells were lysed in RIPA buffer (Beyotime Biotechnology, Shanghai, China) with protease inhibitors. The lysates were separated on SDS-polyacrylamide gels and transferred to 0.22 μm PVDF membranes. The membranes were incubated with 5% skim milk for 2 h at RT and incubated with the primary antibodies at 4 °C overnight. Primary antibodies against the following molecules were used at the following dilutions: PCGF1 (1:1000; ab183499, Abcam), CD133 (1:1000; ab19898, Abcam), SOX2 (1:1000; ab97959, Abcam), Oct4 (1:1000; ab18976, Abcam), cleaved PARP1 (1:1000; ab32064, Abcam), H3K4me3 (1:1000; CST#9751, CST), H3K27me3 (1:1000; CST#9733, CST), H3K9me3 (1:1000; CST#13969, CST), H3K9/K14ac (1:1000; CST#9677, CST), H3K18ac (1:1000; CST#9675P, CST), H3 (1:1000; CST#4499, CST), and β-actin (1:1000; HC201, TransGen Biotech). The membranes were incubated with HRP-conjugated goat anti-mouse (1:5000; HS201-01, TransGen Biotech) or goat anti-rabbit (1:5000; HS101-01, TransGen Biotech) secondary antibodies for 1 h at room temperature after washing. Protein expression was detected with Immobilon™ Western Chemiluminescent HRP Substrate (Millipore). The protein bands were analysed using ImageJ software (64-bit).

The GSCs were lysed with RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing protease and phosphatase inhibitors for 30 min at 4 °C. Then the supernatants were collected after centrifugation at 12,000 rpm at 4 °C for 15 min. The supernatants were mixed with loading buffer (Boster, Wuhan, China), and equal amounts of protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and then transferred to PVDF membranes. The PVDF membranes were blocked and incubated with the primary antibodies at 4 °C overnight. The primary antibodies were used at the following dilutions: rabbit anti-β-actin (1:2000; #4970, CST), mouse anti-β-III tubulin (1:1000; #4466, CST), rabbit anti-GFAP (1:1000; BA0056, Boster), mouse anti-sox2 (1:1000; ab171380, Abcam), rabbit anti-CD133 (1:1000; ab19898, Abcam), rabbit anti-H3 (1:1000; #4499, CST), rabbit anti-OCT4 (1:1000; ab18976, Abcam), rabbit anti-acetyl-H3 (1:1000; #06-599, Millipore), rabbit anti-H3K27me3 (1:1000; #9733, CST), mouse anti-H3K9me3 (1:1000; #5327, CST), rabbit anti-EZH2 (1:1000; #5246, CST). After washing with TBST, the membranes were incubated with HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies at room temperature. The antibody labeling was detected using an enhanced chemiluminescence reagent (Merck Millipore). The protein bands were analyzed using ImageJ.

Peripheral blood samples (0.5 ml) were collected from the retro‐orbital venous plexus at baseline (0) 1, 3, 7, 14, 21, and 28 days after TBI and diluted with PBS. Peripheral blood mononuclear cells were isolated via density‐gradient centrifugation using Ficoll‐Paque Plus (Chuanye, Tianjin, China). The isolated cells were washed twice with PBS and resuspended in 200 μl of PBS supplemented with 0.5% of BSA and 2 mmol/L of EDTA. EPCs in the peripheral blood were evaluated by staining with PE‐conjugated CD34 monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and purified CD133 primary antibody (Abcam Cat# ab19898 Lot# RRID:AB_470302) conjugated with FITC (Abcam Cat# ab6717 Lot# RRID:AB_955238), followed by the detection via flow cytometry (HMS NERCE FACSCalibur Flow Cytometer Resource, RRID:SCR_000879). The isotype‐matched IgG was used as a control.