Bcl 2

Cells were treated with leflunomide for 72 h, then cells were collected, after that proteins were extracted by RIPA Lysis Buffer which contained 1% Phenyl methane sulfonyl fluoride (PMSF), stockpiled at −80°C. The proteins were separated by SDS-PAGE, subsequently, transfered onto PVDF membranes (Millipore, USA). The PVDF membranes were blocked with 5% BSA for 2 h, and incubated with a primary antibody against human Tubulin (1:1000, Beyotime), LC3B (1:1000, Cell Signaling), CDK2 (1:1000, Cell Signaling), CyclinA2 (1:1000, Cell Signaling), DHODH (1:1000, Cell Signaling), BCL-2 (1:1000, Cell Signaling), Caspase-9 (1:1000, Cell Signaling), Caspase-3 (1:1000, Cell Signaling), pT172-AMPK (1:800, Cell Signaling), AMPK (1:800, Cell Signaling), pSer555-Ulk (1:1000, Cell Signaling), Ulk (1:1000, Cell Signaling), pThr 183/186-JNK (1:2000, Abcam), JNK (1:1000, Abcam), BCL-2 (1:1000, Cell Signaling) and Beclin1 (1:1000, Cell Signaling) at 4°C overnight. Then incubated with homologous secondary antibodies HRP-labeled goat anti-rabbit IgG (H+L) (1:2000, Beyotime) or goat anti-mouse IgG (H+L) (1:2000, Beyotime) for 2 h. Finally, the signal was captured by the ECL reagent (Beyotime) and visualized by Western blotting detection instruments (Clinx Science).

10% tissue homogenates from the frontal cortex and hippocampus were prepared in lysis buffer, centrifuged at 13,500 g for 30 minutes at 4°C. Protein concentrations of each sample were determined by the BCA protein assay kit with BSA standards. 60 μg of total proteins were separated on 10% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were incubated for 1 hour with 5% dry skim milk in TBST buffer at room temperature to block nonspecific binding. Membranes were then incubated with primary antibodies against caspase-3 at a dilution of 1:1000 (the rabbit anti-mouse cleaved caspase-3 mAb, Cell Signaling Technology, USA), Bcl-2 at 1:1000 (the rabbit anti-mouse Bcl-2 mAb, Cell Signaling Technology, USA), Mfn2 at 1:1000 (the rabbit anti-mouse mitofusin-2 mAb, Cell Signaling Technology, USA), Drp1 at 1:1000 (the rabbit anti-mouse Drp1 mAb, Cell Signaling Technology, USA), β-actin at 1:2000 (ZS-Bio, China) overnight at 4°C. Subsequently, membranes were incubated with alkaline-phosphatase-conjugated secondary antibodies for 1 hour at room temperature. Bands were visualized using the chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate in the presence of nitroblue tetrazolium (SIGMA, USA). The band density was scanned and analyzed using the Odyssey Two-Color Infrared Imaging System (LI-COR, USA).

A1874 was obtained from Hanxiang BioTech (Shanghai, China). Puromycin, JQ1, CPI203, I-BET151, N-acetyl-cysteine (NAC) and pifithrin-α, polybrene and CCK-8 were purchased from Sigma-Aldrich (St. Louis, MO). All cell culture reagents were provided by Hyclone Co. (Logan, UT). Antibodies for c-Myc (#9402), Cyclin D1 (#2922), BRD4 (#13440), Bcl-2 (#15707), Erk1/2 (#9102), p53 (#9282), cleaved-caspase-3 (#9664), cleaved-caspase-9 (#20750), cleaved-poly (ADP-ribose) polymerase (PARP) (#5625), Bcl-2 (#15707) and β-tubulin (#15115) were purchased from Cell Signaling Tech (Beverly, MA). Caspase inhibitors, z-VAD-fmk and z-DEVD-fmk, were provided by Thermo-Fisher (Shanghai, China). Lipofectamine 2000, TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling), Annexin V and propidium iodide (PI) were purchased from Thermo-Fisher Invitrogen (Carlsbad, CA). The BrdU ELISA kit was provided by Roche Diagnostics (Basel, Switzerland).

Briefly, total protein from tumor samples and cell lines was isolated using RIPA lysis buffer (Servicebio, China), and protein concentration was measured by BCA Protein assay kit (Thermo Fisher Scientific, UAS, 1:1000). Western blot analysis of CRHBP (Thermo Fisher Scientific, UAS, 1:1000), P-AKT (Cell Signaling Technology, USA, 1:1000), AKT (Cell Signaling Technology, USA, 1:1000), p-p65 (Cell Signaling Technology, USA, 1:1000), p65 (Cell Signaling Technology, USA, 1:1000), P-IκBα (Cell Signaling Technology, USA, 1:1000), IκBα (Cell Signaling Technology, USA, 1:1000), p-p53 (Cell Signaling Technology, USA, 1:1000), p53 (Cell Signaling Technology, USA, 1:1000), Bax (Cell Signaling Technology, USA, 1:1000), Bcl2 (Cell Signaling Technology, USA, 1:1000), Bcl-2 (Cell Signaling Technology, USA, 1:1000), Bcl-xl (Cell Signaling Technology, USA, 1:1000), Cytochrome c (Cell Signaling Technology, USA, 1:1000), β-actin (Abcam, Cambridge, UK, 1:5000) and GAPDH (Cell Signaling Technology, USA, 1:5000) was according to standard protocols. The blots were scanned with two-color infrared imaging system (Odyssey, LI-COR, USA) after incubating with goat-anti-rabbit secondary body (LI-COR, USA, 1:2000), and analyzed by image J software.

HCT116 cells were harvested, lysed on ice in RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China) and boiled. Protein concentration was measured by BCA protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). The protein samples were separated by SDS-PAGE and transferred to the membrane (Pall, New York, NY, USA). Afterwards, the membrane was immunoblotted with the corresponding primary antibody (C-parp (#5625), C-cas9 (#9505), C-cas3 (#9661), Bcl-2 (#4223), Bax (#5023), SHP2 (#3752), P-Erk1/2 (#9101) and Erk1/2 (#9102), 1:1000, Cell Signaling Technology, Danvers, MA, USA; Tubulin (#ET1602-4), 1:5000, HUABIO, Hangzhou, China) and the appropriate HRP-conjugated secondary antibody, and determined with ECL detection reagent (Thermo Scientific, Waltham, MA, USA).