Anti glut1

Paraffin-embedded samples of keloid and normal tissue were cut and mounted on coated slides. After de-paraffinization in xylene and rehydration with graded alcohol solutions, antigen retrieval was performed in sodium citrate buffer (100 °C, 20 min). The Novolink Polymer Detection System (RE7150-K, Leica, Wetzlar, Germany) was carried out according to the manufacturer’s introduction. The sections were neutralized with endogenous peroxidase using a Peroxidase Block for 5 min, blocked by incubation with Protein Block (RE7102, Leica, Wetzlar, Germany) for 5 min, then incubated overnight at 4 °C with mouse monoclonal antibody anti-GLUT-1 (1:100 dilution, sc3772282, Santa Cruz, CA, USA). After rinsing by phosphate buffered saline (PBS) (11-223-1M, Biological Industries, Cromwell, CT, USA), the slides were incubated with Novolink Polymer for 30 min. Afterwards, the sections were developed with 3,3′-diaminobenzidine (DAB) working solution, counterstained with hematoxylin for 5 min, dehydrated, and mounted by mounting medium. Finally, 6 view fields were randomly selected on each section and observed under a microscope (Olympus, Tokyo, Japan) to determine the expression.

Cells were lysed (5 × 10⁶) CLL in RIPA buffer and 30 μg proteins separated by SDS–polyacrylamide gel electrophoresis. Proteins were transferred to Immobilon-P membrane (Millipore Corp, Bedford, MA, USA) and probed with primary antibody overnight at 4 °C (anti-HIF-1α—BD Biosciences, anti-GLUT1- Santa Cruz Biotechnology (Dallas, TX, USA), anti-VEGF- Abcam (Cambridge, UK), anti-LDHA-Abcam, anti-β-actin-Sigma-Aldrich) followed by secondary anti rabbit (Sigma-Aldrich) or anti mouse conjugated with horseradish peroxidase (Sigma-Aldrich). Signal was developed using Supersignal West Pico Chemiluminescent substrate (Pierce, Northumberland, UK) and detected by exposure to Kodak Xomat imaging film (Sigma-Aldrich). Films were developed using an AGFA CURIX 60 (Agfa, Mortsel, Belgium).

Primary astrocytes were seeded at 5 × 10⁵ cells/well in 6-well plates, allowed to attach overnight, and then incubated in DMEM containing 1 μg/mL adiponectin for 24 h. The cells were washed twice with PBS, followed by scraping and resuspension of the cell pellet in RIPA lysis buffer containing protease inhibitors and centrifugation to remove debris, unbroken cells, and cellular nuclei. Protein content was determined by Bradford’s method using bovine serum albumin as a standard. Samples containing 10 µg of total protein were separated using 12% sodium dodecyl sulfate acrylamide gels. After electrophoresis, proteins were transferred to a nitrocellulose membrane and then blocked with 5% skim milk in TBS (Tris-buffered saline, 0.1% Tween20) buffer for 2 h and incubated in the primary antibodies, including anti-GFAP (1:1000 dilution, Sigma-Aldrich, USA), anti-Iba-1 (1:1000 dilution, Wako, Osaka, Japan), anti-Glut1 (1:500 dilution, Santa Cruz Biotechnology, Dallas, TX, USA), anti-phosphorylated (p)AMPK, anti-total AMPK (1:1000 dilution, Cell Signaling, Danvers, MA, USA), and anti-β-actin (1:10,000 dilution, Sigma-Aldrich, USA).

Human anti-cleaved PARP and anti-flag antibodies were obtained from Cell Signaling Technology. Anti-tubulin was procured from Bioworld Company. Anti-GLUT1 and anti-LDHA antibodies were purchased from Santa Cruz Biotechnology. Anti-SUN2 (HPA001209) and anti-Sirt5 (HPA021798) antibodies were obtained from Sigma-Aldrich.