Lc3bi 2

After SAH and sham rats were sacrificed at 24 h following endovascular perforation, brain tissue samples from around the basal cortical area on the injured side were obtained for assessment. Western blotting was performed as previously described (Pariente et al., 2016 (link)). Proteins were extracted from the brain tissue samples with RIPA buffer (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The primary antibodies used were antibodies against MST1 (Cell Signaling Technology, Danvers, MA, USA, CST#3682), cl-MST1 (Cell Signaling Technology, CST#3682), active Caspase 3 (Abcam, ab49822), Bcl-2 (Cell Signaling Technology, CST#2876), Bax (Santa Cruz Biotechnology, SC-493), Beclin 1 (Proteintech, 11306-1-AP), and LC3B-I/II (Cell Signaling Technology, CST#4108).

The damaged hippocampal region tissues were separated with RIPA buffer (Beyotime, China) on ice and homogenized to extract protein. Forty μg proteins were separated with 12% SDS-PAGE (Bio-Rad, China) and transferred to PVDF membranes (EMD Millipore). After blocking with 5% milk for 1.5 h, the membranes were incubated with appropriate primary antibodies diluted with 5% BSA for overnight at 4 °C. The primary antibodies consisted of phospho-PPARγ (ser273) (1:1000, bs-4888R, Bioss, China), LC3BI/II (1:1000, 4108, Cell signaling technology, China), phospho-NF-κB p65 (1:800, bs-230303R, Bioss, China), GAPDH (1:1000, bs-0755R, Bioss, China). After washing with TBS-0.01% Tween 20 for 3 times (10 min/wash), the secondary antibody Goat Anti-rabbit lgG/HRP (1:1000, bs-0295G-HRP, Bioss, China) was cultured with the membranes for 2 h at 25 °C. After washing, the signals were visualized using enhanced chemiluminescence reagent (D085075, Bio-Rad, China).

Alteration of specific proteins post-treatment was determined by seeding cells of the GSC culture GS257 in culture flasks with 225nM Obatoclax, 1 μM SAHA and the combination. After 24 hours, the cells were irradiated with 3Gy. At 24 and 48 hours post-RTx, the cells were harvested, washed with PBS and collected in a 1% Triton-X100 (Sigma-Aldrich) buffer for protein isolation. Protein concentrations were measured with the BCA Protein Assay Reagent Kit (Thermo Fisher Scientific, MA, USA). Protein separation was performed on a 4-15% pre-casted gel (Bio-Rad, CA, USA), and blotted onto a PVDF membrane (Immobilon-P, Millipore, MA, USA) using the Mini-Protean Tetra Cell system (Bio-Rad). After blocking the membranes with 5% non-fatty milk solution for 30 minutes at room temperature, the blots were incubated with primary antibodies against Bcl-XL, LC3BI/II, Mcl-1 (1:375, Cell Signalling, MA, USA) and anti-β-actin (1:5,000, Millipore) in 5% BSA/TBS-T overnight. Membranes were washed with TBS-T 0.2% and incubated with secondary antibodies, anti-rabbit-HRP and anti-mouse-HRP (1:2000, Dako Denmark A/S, Denmark) 1½h at room temperature and detected by chemiluminescence using the Pierce ECL substrate (Thermo Fisher Scientific) and the ChemiDoc MP system (BioRad). The data were analyzed using the ImageLab software (Bio-rad).

After treatment of different doses of TFP, 100 nM bafilomycin A1, 2.5 μM rapamycin, 4 Gy radiation at a dose rate of 1.8 Gy/min using a linear accelerator (Primus Hi; Siemens Medical Instruments; Erlangen, Germany) or 5 μM TFP for 24 h before receiving one dose of 4 Gy, whole-cell protein extracts (20-50 mg) were prepared using a radioimmunoprecipitation assay buffer (RIPA; Thermo Fisher Scientific) supplemented with protease inhibitor cocktail (Cell Signaling Technology; Beverly, MA, USA). Proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. Membranes were blocked with 5% skimmed milk in Tris-buffered saline containing 0.1% Tween-20, and subsequently incubated with primary and indicated secondary antibodies (Thermo Fisher Scientific). Proteins on western blots where visualized using the Chemiluminescent Reagents Kit (Millipore, Billerica, MA, USA). Chemiluminescent signals were detected with the ChemiDoc XRS+ (Bio-Rad, Hercules, CA, USA) and quantified using Image Lab 3.0 software (Bio-Rad). The following primary antibodies were used for western blotting: beta-tubulin, LC3BI/II, phospho-histone H2A.X (Ser139; also known as γ-H2A.X), P62 and survivin (Cell Signaling Technology; Beverly, MA, USA); GAPDH, BRCA1, BRCA2 and Rad51 were purchased from Santa Cruz (Dallas, TX, USA).

HTO was obtained from Medalchemy (Alicante, Spain). The RPMI medium, triolein, RNaseA and propidium iodide were purchased from Sigma-Aldrich (St. Louis, USA), with penicillin-streptomycin purchased from Biowest (Nuaillé, France) and the fetal bovine serum (FBS) from Biosera (Boussens, France). The recombinant human EGF was obtained from R&D systems (Minneapolis, USA), while sodium orthovanadate was from Sigma-Aldrich (St. Louis, USA) and the protease inhibitors were from Roche (Roche, Basel, Switzerland). NBD-sphingomyelin and NBD-glucosylceramide were purchased from Larodan (Solna, Sweden), and NBD-ceramide from Invitrogen, Molecular Probes. The antibodies used to probe Western blots were raised against LC3BI-II, ERK, phospho-ERK (p-ERK), Akt and phospho-Akt (p-Akt S473), and they were purchased from Cell Signaling (Danvers, MA, USA). The antibody against α tubulin was purchased from Sigma-Aldrich (St. Louis, USA) and the antibodies against EGFR and p-EGFR (Y1068) were from Abcam (Cambridge, UK). The anti-EGFR antibody 930 used to stain the cell surface and to assess internalization was kindly provided by Genentech (San Francisco, USA).

The prostate cancer cell lines LNCaP and PC3 and HCT116 colon cancer cells were obtained from Georgetown University (GU) Lombardi Comprehensive Cancer Center (LCCC) cell culture repository and cultured in DMEM (Invitrogen, Carlsbad, CA) medium supplemented with 10% fetal bovine serum (FBS), 2mM-glutamine, 25ug/ml of gentamicin (Invitrogen) and incubated at 37^◦C with 5% CO₂. Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from Lonza (Allendale, NJ) and cultured as described [49 (link)]. The following primary antibodies: SIRT1, LC3B-I/II, and GAPDH antibodies were purchased from (Cell Signaling Technology Inc., Danvers, MA). Secondary antibodies conjugated with horseradish peroxidase were from Jackson ImmunoResearch (West Grove, PA).