Cleaved caspase 3

Total proteins from tissues and cells were harvested, washed, and lysed in radioimmunoprecipitation buffer (RIPA) buffer. The protein concentration of each purified exosome sample was determined using a bicinchoninic acid (BCA) protein assay kit (CWBIO). Equal amounts of tissue or cell lysates were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. After being blocked with 5% skim milk for 1 h, the membranes were incubated with primary antibodies and horseradish peroxidase (HRP)-conjugated secondary antibodies and detected using an enhanced chemiluminescent (ECL) substrate detection system. The primary antibodies used in the experiments were as follows: CD9 (1:500, Bioworld Technology, USA), CD63 (1:500, Bioworld Technology, USA), Bax (1:500, Bioworld Technology, USA), Bcl2 (1:500, Bioworld Technology, USA), SIRT1 (1:500, Bioworld Technology, USA), PCNA (1:500, CST, USA), 14-3-3ζ (1:500, Bioworld Technology, USA), TNF-α (1:500, Bioworld Technology, USA), Cleaved caspase3 (1:400, Bioworld, USA) GAPDH (1:2000, CWBIO, China), β-actin (1:2000, CWBIO, China). Primary antibodies were incubated overnight at 4° C. The HRP-conjugated goat anti-rabbit and goat anti-mouse secondary antibodies (1:2000, CWBIO, China) were incubated 2 hours at 37° C.

Min6 cells were plated in six-well dishes with overnight culture and then incubated with 10 μmol/L tested compounds and 300 μmol/L PA for 12 h. Total proteins of cells were lysed with RIPA lysis buffer (Boster, China) and the protein lysis was separated by SDS-PAGE and transferred from gel to PVDF membrane (Millipore, USA). Then, the membranes were blocked, stained overnight with corresponding primary antibodies and then with second antibody for 1 h. The signals of detected proteins were measured by Chemidoc Imaging System (BIO-RAD, USA). The antibody used were as follows: TXNIP (Abcam, ab188865), NLRP3 (Wanlei, Wuhan, China; WL02635), Cleaved caspase 1 (Wanlei, Wuhan, China; WL03450), Cleaved caspase 3 (CST, #9664s), GAPDH (Bioworld, Nanjing, China; AP0063).

Nuclear and cytosolic proteins were extracted from liver tissues with a protein extraction kit (KeyGen Biotech, Nanjing, China), and cells were lysed with RIPA buffer. Equal amounts of protein from each sample were separated by 10–15% SDS-PAGE (Bio-Rad, Hercules, USA). Blots were incubated overnight at 4 °C with the following primary antibodies: SIRT1 and p66shc (Abcam Ltd, Cambridge, UK); MnSOD, Bcl-xL and cleaved caspase-3 (Bioworld Technology, Inc., St Louis Park, MN, USA); and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The blots were then immunostained with secondary antibodies at 37 °C. The membranes were exposed to enhanced chemiluminescence-plus reagents (Beyotime Institute of Biotechnology). The images were captured using a BioSpectrum-410 multispectral imaging system and analyzed with Gel-Pro Analyzer (Version 5.0; Media Cybernetics, Rockville, MD, USA).

α-Hederin of purity over 99% was provided by Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China). NF-κB specific inhibitor pyrrolidine dithiocarbamate (PDTC), JAK2/STAT3 signaling specific inhibitor AG490, and ERK specific inhibitor U0126 were obtained from Selleck Chemicals (Houston, TX, USA). Dimethyl sulfoxide (DMSO) was used to dissolve the above compounds for experiments, and single treatment with DMSO was used as negative control. Recombinant human IL-6 cytokine was obtained from Solarbio Life Science (Beijing, China). The primary antibodies used for Western blot analyses against cyclin B1, CDK1, Bcl-2, Bax, Cyt c, cleaved-caspase-9, cleaved-caspase-3, cleaved-caspase-8, cleaved-PARP, NF-κB(p65), p-IκBα, IκBα, p-IKKα, IKKα, p-ERK, ERK, COX IV, lamin B1, and GAPDH were purchased from Bioworld Technology, Inc. (MN, USA).