Anti chop

Protein was extracted from the cultured cells using RIPA buffer. A total of 30 μg proteins were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, proteins were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in TBST containing 5% skimmed milk, and incubated with the primary antibodies including: anti-GRP78 (1:1000; Sigma Aldrich), anti-PERK (1:1000; Sigma Aldrich), anti-p-PERK (1:500; Sigma Aldrich), anti-eIF2α (1:1000; Abcam, Cambridge, UK), anti-p-eIF2α (1:500; Abcam), anti-ATF4 (1:1000; Sigma Aldrich), anti-CHOP (1:500; Sigma Aldrich), anti-SIRT1 (1:1000; CST, Beverly, USA), anti-Bcl-2 (1:1000; Proteintech, Wuhan, China), Bax (1:1000; Proteintech), anti-Mcl-1(1:1000; Sigma), anti-caspase 3 (1:1000; CST), anti-caspase 9 (1:1000; CST), anti-cleaved caspase 3 (1:500; Sigma Aldrich), and anti-β-actin (1:1000; Origene, Rockville, USA), followed by incubation with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies. Finally, chemiluminescent substrate (Beyotime) was used for signal visualization and detection. The density of each band was normalized to its respective loading control (β-actin). The immunoblots were quantified by densitometric analysis.

The tissue sections were deparaffinized and were then rehydrated and blocked with 3% H2O2 in methanol followed by antigen retrieval in a microwave in 10 mM citrate buffer, pH 6.0. Tissue sections were treated with universal blocking solution (BioGenex, HK085–5K) for 10 min, and then incubated overnight at 4°C with the following primary antibodies: anti-XBP1 (Santa Cruz Biotechnology), anti-CHOP (Sigma-Aldrich,), anti-BiP (Sigma-Aldrich,), anti-pro/active-CASP3/caspase 3 (Novus Biologicals) and anti-NFκB (Abcam). A secondary biotinylated anti-immunoglobulin followed by horseradish peroxidase-conjugated streptavidin (BioGenex, HK330–5K) was used according to the manufacturer's instructions. 3-amino-9-ethyl-carbazole (BioGenex, HK092–5K) in acetate buffer containing 0.05% H2O2 was used as the substrate. The sections were counterstained with hematoxylin. The primary antibody was replaced by nonimmune serum for negative control slides. Image analysis and cell counting was performed using the Nikon software, Nis Elements 3.0.
Positive signals were distinguished from cytoplasmic staining and were quantified using automated image analysis from different fields at 40X magnification. All photomicrographs were taken with the same settings for exposure time and light. The results are represented in bar graphs as the percentage of positive cells per field.

Cell lysates were prepared by RIPA buffer (1XPBS, 1% nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS, pH 8.0). Protein concentrations were determined by bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, US). After equal amounts of proteins were loaded to the gels, proteins were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes (EMD Millipore, Thermo Fisher Scientific, US). Following the classic immunoblotting steps (blocking, incubating with primary and secondary antibodies), chemiluminescence signals were detected using Clarity ECL substrate solution (Bio-Rad, US) by Fusion-FX7 (Vilber Lourmat, Thermo Fisher Scientific, US). Monoclonal antibodies used in this study were anti-LC3 (CST-12741, USA), anti-Atg-5/12 (CST-12994, USA), anti-Atg-7 (CST-8558, US), anti-actin (Sigma-Aldrich-A5316, UK), anti-caspase-3 (CST-9665, US), Anti-CHOP (CST-2895, UK). The experiments were repeated three times independently; with one representative result shown.