β actin

Peritumoral edema tissue was ground in liquid nitrogen and dissolved in a RIPA Lysis Buffer (Beyotime Institute of Biotechnology), which contained the protease inhibitor phenylmethanesulfonyl fluoride. The total protein was extracted from the tissue, and the protein concentration was determined using the BCA Protein Assay Kit (catalog no. P0012; Beyotime Institute of Biotechnology) and stored at −80°C for additional analysis. Equal amounts of protein (>20 µg) from each group were loaded and resolved using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (0.45 µm; EMD Millipore, Boston, MA, USA). The membranes were blocked with 5% non-fat milk in Tris-buffered saline, and subsequently incubated with rabbit anti-rat MMP2 and rabbit anti-rat NKCC1 antibodies at 4°C overnight. The membranes were incubated with goat anti-rabbit secondary antibody (monoclonal; dilution, 1:2,000; catalog no. TA130024; OriGene Technologies, Inc.) for 2 h at room temperature. The optical density of bands was determined using ImageJ software (version 1.48; National Institutes of Health, Bethesda, MA, USA) and the data were quantified by normalization to the density of β-actin (OriGene Technologies, Inc.).

Temozolomide (TMZ) was purchased from Aladdin Reagent Company (http://www.aladdin-e.com). Other reagents were purchased from Beyotime Institute of Biotechnology. Primary antibodies: EGFR, cat no. 18986-1-AP, 1:1,000, Proteintech Group, Inc., Chicago, IL, USA; STAT3, cat no. 9139S, 1:1,000, Cell Signaling Technology, Inc., Danvers, MA, USA; p-STAT3, cat no. 9145S, 1:1,000, Cell Signaling Technology, Inc.; HIF-1A, cat no. 14179S, 1:1,000, Cell Signaling Technology, Inc.; VEGF-A, cat no. 19003-1-AP, 1:1,000, Proteintech Group, Inc.; Bax, cat no. 50599-2-Ig, 1:1,000, Proteintech Group, Inc.; Bcl-2, cat no. 12789-1-AP, 1:1,000, Proteintech Group, Inc.; MGMT, cat no. A-1010-050, 1:1,000, Epigentek, Farmingdale, NY, USA; β-actin, cat no. TA-09, 1:1,000, OriGene Technologies, Inc., Rockville, MD, USA). HRP-conjugated secondary antibodies: HRP-conjugated Affinipure Goat anti-mouse IgG, cat no. SA00001-1, 1:2,000, Proteintech Group, Inc.; HRP-conjugated Affinipure goat anti-rabbit IgG, cat no. SA00001-2, 1:2,000, Proteintech Group, Inc.

The Ca9-22 cells were lysed in ice-cold RIPA buffer (cat. no. R0020; Beijing Solarbio Science & Technology Co. Ltd.) with protease inhibitor for 30 min and centrifuged at 12,000 × g for 20 min at 4°C. The protein concentration was determined by the Bradford method (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) as the standard. Each protein lysate (40 µg) were separated on 10% SDS-PAGE gels and transferred to a polyvinyl difluoride membrane (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The membrane was blocked with 5% skim milk (Santa Cruz Biotechnology, Inc., Dallas, TX, USA) [PBS was diluted to obtain 5% milk seal solution (5 g/100 ml)] for 40 min at room temperature, and then incubated with the primary (4°C overnight) and secondary antibodies (secondary goat anti-rabbit (cat. no TA130015; 1:000) or anti-mouse antibody (cat. no TA100015; 1:000); OriGene Technologies, Inc., Rockville, MD, USA) for 1 h at room temperature. The primary antibodies were USP22 (1:1,000; Abcam; cat. no. ab71732), Survivin (cat. no. NB500-201; Novus Biologicals; 1:2,000), Aurora-B (cat. no AMI-1; 1:000; Transduction Laboratories), Cyclin B (cat. no. 610219), p21 (cat. no. clone 70) (1:1,000; Transduction Laboratories) and β-actin (1:2,000, OriGene Technologies, Inc.; cat. no TA-09).

Western blot analysis was performed following protein extraction from HUVECs as described previously (17 (link)). Antibodies against PBX3 (cat. no. ab56239, 1:1,000 at 4°C overnight) and HMGB1 (cat. no. ab79823, 1:5,000 at 4°C overnight) were purchased from Abcam (Cambridge, UK) and β-actin was purchased from OriGene Technologies, Inc. (Beijing, China). Blots were washed and incubated with a hydrogen peroxidase-conjugated secondary antibody (ab6728 or ab6721, Abcam, 1:5,000 for 1 h at room temperature), and chemiluminescence was detected using an enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.) followed by exposure with a Tanon 5200 Multi Chemiluminescent System.