Ab74290

The tested compounds were provided by Dongdong Sun (Nanjing University of Chinese Medicine). For the treatment, the compounds were dissolved in culture medium and DMSO [with DMSO less than 0.1%(v/v)]. HCT116 colorectal cancer cells were supplied from the cell bank of the Chinese Academy of Sciences (Shanghai, China). RPMI 1640 culture medium was provided by Biological Industries (Beit‐Haemek). TUNEL apoptotic commercial kit was produced by Beyotime. Annexin V‐PI kit was purchased from KeyGEN. α‐ketoglutarate as obtained from Jinglai Biotechnology. The commercial kits for ATP, glutamine, glutamate and glutathione were provided by Jiancheng Biotechnology. The ASCT2 inhibitor GPNA was produced by Sigma‐Aldrich. Pifithrin‐α, the selective inhibitor for P53, was obtained from APExBIO. ASCT2 (#ab84903, 1:1000), P53 (#ab26, 1:1000), caspase‐3 (#ab197202, 1:1000), caspase‐7 (#ab69540, 1:1000), cleaved‐caspase‐3 (#ab2302, 1:1000), cleaved‐caspase‐7 (#ab2323, 1:1000), cleaved‐PARP (#ab32064, 1:1000), PARP (ab74290, 1:1000) and survivin (ab76424) were purchased from Abcam.

Protein was extracted using protein extraction kit (KeyGEN BioTECH), and protein concentration was determined by BCA Protein Assay Kit (KeyGEN BioTECH). 30 μg of protein was separated by SDS‐PAGE and transferred onto PVGF membranes, which were followed by incubating with 5% non‐fat milk for 1.5 hours at room temperature. Afterwards, membranes were incubated with the primary antibodies against six2 (ab132611), ALDH1 (ab23375), Nanog (ab21624), cleaved caspase 3 (ab2302), caspase 3 (ab13847), cleaved PARP (ab32064), PARP (ab74290), which were purchased from Abcam. Primary antibody against β‐actin (cat # AF0003) was purchased from Beyotime. After incubating with primary antibodies, blots were washed and incubated with a secondary peroxidase‐conjugated antibody (KeyGEN BioTECH), and chemiluminescence was detected using an enhanced chemiluminescence kit (ThermoFisher Scientific) followed by exposure in Tanon 5200 (Tanon).

Protein extracts (10 μg) prepared from LCLs or B lymphocytes were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, Wisconsin, USA). The membranes were blocked with 5% milk in Tris-buffered saline (pH 7.6) containing 0.1% Tween 20 and incubated overnight at 4°C with specific primary antibodies against the following proteins: TNF receptor-associated factor 1 (TRAF1, 1 : 1000, ab203316, abcam, Cambridge, UK), IκB kinase (IKK)-α (1 : 10000, ab109749, abcam), NF-κβ-inducing kinase (NIK, 1 : 500, ab203568, abcam), P52 (1 : 2000, ab129097, abcam), pro-caspase 3 (1 : 1000, ab32150, abcam), caspase 3 (1 : 500, ab49822, abcam), poly (ADP-ribose) polymerase (PARP, 1 : 2000, ab74290, abcam), and β-actin (1 : 5000, 66009-1-Ig, Proteintech, USA). After three washes with PBS/Tween 20, the membranes were incubated with horseradish peroxidase-conjugated secondary goat antirabbit IgG (1 : 20000, PAB160011, Bioswamp) for 2 h at room temperature. Protein bands were visualized by enhanced chemiluminescence color detection (Tanon-5200, TANON, Shanghai, China) and analyzed using AlphaEase FC gel image analysis software.

Cell protein was collected by RIPA lysis buffer (KeyGEN, Nanjing, China) containing 1 nM PMSF (Biotool, Houston, TX, USA). 20 μg protein per well was electrophorsed in 10% SDS-PAGE gels and then transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA), and incubated with different primary antibodies, including GALNT3 (16716–1-AP, Proteintech, China), caspase3 (ab13847, Abcam, the UK), cleaved caspase3 (ab13585, Abcam, the UK), PARP (ab74290, Abcam, the UK), cleaved PARP (ab4830, Abcam, the UK), MUC1 (ab15481, Abcam, the UK), PI3K-p110α (21890–1-AP, Proteintech, China), p-AKT 308 (AP3743a, Abgent, China), p-AKT 473 (AP3434a, Abgent, China), AKT (ab8805, Abcam, the UK), NF-κB (AP50006, Abgent, China) and GAPDH (AP7873a, Abgent, China), at 4 °C overnight. The membrane was treated with anti-rabbit IgG at 37 °C for 2 h. All bands were detected by an ECL Western blot kit (Thermo Fisher Scientific, USA) and analyzed by Lab Works (TM ver4.6, UVP, Bio Imaging Systems, NY, USA). GAPDH was used as control.

Following the obtaining of proteins by Radio-Immunoprecipitation Assay (RIPA) lysis and extraction buffer (Thermo Fisher Scientific, Waltham, MA, USA), 40 μg proteins were split on 10% sodium dodecyl sulfate–polyacrylamide gel (Invitrogen) for 2 h. 5% skim milk (Thermo Fisher Scientific) was implemented to block the non-specific binding signals after the transferring of proteins onto the polyvinylidene fluoride membranes (Thermo Fisher Scientific). Then membranes were incubated with primary antibodies: anti-CREBRF (Abcam, Cambridge, UK, ab26262, 1:1000), anti-poly-ADP-ribose polymerase (anti-PARP; Abcam, ab74290, 1:1000), anti-Cleaved PARP (Abcam, ab30264, 1:1000), anti-Cleaved caspase-3 (Abcam, ab2302, 1:1000) and anti-β-actin (Abcam, ab8227, 1:3000) for 3 h at room temperature. Subsequently, the secondary antibody (Abcam, ab205718, 1:5000) was exploited to bind to primary antibodies. 1 h later, the detection of combined signals was administered by the enhanced chemiluminescence reagent (Abcam). Ultimately, ImageLab software version 4.1 (Bio-Rad Laboratories, Hercules, CA, USA) was used for image acquisition and densitometric analysis previously [22 (link)].

After drying at room temperature, slices were fixed by pre-cold acetone for 6 min, washed by 0.01 m PBS for three times, incubated with formamide/twice sodium citrate hybrid solution at 65 °C for 2 h, incubated with 0.3% triton for 30 min, washed by PBS for 3 times, washed by 2 m HCl at 37 °C for 30 min, by 0.1 mol/L boric acid buffer (pH = 8.0) twice, by 0.01 m PBS twice, and then blocked by 10% goat serum (Shanghai Sangon Biotechnology Co. Ltd., Shanghai, China) for 1 h at 37 °C. And then, slices were incubated with GRP78 (Abcam, ab21685, rabbit, 1: 2000) overnight at 4 °C. After incubation, slices were washed by PBS and incubated with FITC-labeled IgG (Abcam, ab74290, 1:2000) for 45 min at room temperature. After that, slices were mounted by anti-fluorescence quenching agent and observed under a confocal microscope (Leica Microsystems, Mannheim, Germany).