Anti cleaved caspase 3 antibody

For analyses of endothelial and perivascular cells, tumor tissues containing the total areas of CD31 (clone DIA-310, Dianova, Germany) positive endothelial cells and α-smooth muscle actin (α-SMA) (Sigma-Aldrich, Saint Louis, Missouri, USA) positive pericytes were identified by scanning tumor sections under ×10 magnification and were counted in five random fields under ×200 magnification. CD8+ lymphocytes were detected using anti-CD8b antibody (eBioscience). Five random fields were selected from the tumor center, which did not include the edge of the tumor. Hypoxia was identified and quantified using immunostaining for anticarbonic anhydrase (CA)-IX (rabbit monoclonal; dilution, 1:100, Abcam). These data were analyzed using ImageJ (US NIH) and Photoshop (Adobe Systems, San Jose, California, USA) software. Analysis was performed using a laser-scanning confocal microscope (Olympus, FV-1000). Anticleaved caspase-3 antibody (Abcam) was used to assess the cell apoptosis in the tumor tissues, and antiphospho-STAT3 antibody (Abcam) for quantitative assessment of p-STAT3. For the latter, immunostained whole slide tumor sections were scanned on a Hamamatsu NanoZoomer S360 slide scanner. For quantification purpose, whole images were analyzed for at least three representative areas of vital tumor which was defined as region-of-interest.

Dulbecco's modified eagle medium (DMEM) was purchased from Procell Life Science & Technology Co., Ltd (Wuhan, China). Penicillin–streptomycin and fetal bovine serum (FBS) were purchased from Shanghai BasalMedia Technologies Co., Ltd (Shanghai, China). DAB Assay Kit was obtained from ZSGB-Bio Co., Ltd (Beijing, China). Hoechst 33342/PI Assay Kit was obtained from KeyGEN Biotechnology Co., Ltd (Jiangsu, China). CCK-8 Assay Kit, EDU-594 Assay Kit, JC-1 Assay Kit and BCA Protein Assay Kit and p-IRE1α rabbit pAb were purchased from Beyotime Biotechnology Co., Ltd (Shanghai China). Anti-Cleaved Caspase-3 antibody was obtained from Abcam (Cambridge, UK). DDIT3/CHOP rabbit mAb, Caspase-3 rabbit pAb, ATF6 rabbit pAb and BiP/GRP78 rabbit mAb were purchased from ABclonal Biotechnology Co., Ltd (Wuhan, China). p-PERK pAb and PERK pAb were obtained from Thermo Fisher Scientific Co., Ltd (MA, USA).

Liver specimens or cell cultures were lysed in RIPA buffer (Thermo Fisher scientific, Waltham, MA, USA), mixed with proteases inhibitors (Roche), for 30 min at 4°C, and then, centrifuged at 13 000 rpm at 4°C for 10 min. Protein samples were separated using SDS‐PAGE and then, transferred onto PVDF membranes. After blocking, the membranes were incubated with primary antibodies (anti‐PI16 antibody, anti‐β‐actin antibody, anti‐Cleaved caspase‐3 antibody: Abcam, Cambridge, UK; anti‐PARP antibody, anti‐p38 antibody, anti‐p‐p38 antibody, anti‐AKT antibody, anti‐p‐AKT antibody: Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight. Then the membranes were washed with TBST (20 mM Tris‐HCl, 150 mM NaCl, 1% Tween‐20) and incubated with HRP‐conjugated secondary antibodies (1:5000; Abcam, Cambridge, MA, USA) for 1 hour. The protein signals were revealed using enhanced chemiluminescence (Merck Millipore, Billerica, MA, USA). All western blots were conducted at least three times, and the images are representative of consistent results.