Bcl2 associated x (bax)

Total proteins were extracted from frozen hepatocytes and hepatic samples, and then the protein concentrations were measured by BCA protein assay (Solarbio, China). The samples were separated on SDS-PAGE and transferred to a polyvinylidene fluoride membrane. The membrane was blocked with 5% skimmed milk in Tris-buffered saline containing 0.2% Tween-20 at ambient temperature for 1 h, followed by incubation with following antibodies: GAPDH (1:5000; Proteintech, China), ALB (1:2000; Proteintech, China), CYP3A4 (1:2000; Abcam, UK), alpha 1 antitrypsin (AAT, 1:2000; Abcam, UK), cytokeratin 18 (CK-18, 1:2000; Abcam, UK), Bax (1:2,000; Proteintech, China), and Bcl-2 (1:2000; Proteintech, China). The membrane was treated with HRP-conjugated goat anti-rabbit secondary antibody (1:3000; Cell Signaling Technology, USA) for 1 h at room temperature. The protein bands were visualized with an ECL chemiluminescence solution (Millipore, USA) and imaged using the Imaging System (BIO-RAD, USA). Protein expression levels were determined by densitometry using Image J software.

The expression of cleaved caspase-3, Bcl-2, Bax, Fas and FasL was analyzed by Western blotting. β-actin was used as the internal control. Briefly, after total protein was extracted from NP cells with RIPA buffer (Beyotime, China), protein concentration was determined by the BCA assay kit (Beyotime, China). Then, equal protein samples in each group were sequentially subjected to SDS-PAGE and transferred to the PVDF membrane. After the PVDF membranes were blocked with 5% bovine serum albumin (BSA) for 1 h at 37°C, they were incubated overnight at 4°C with corresponding primary antibodies (cleaved caspase-3: Cell Signaling Technology, #9661; Bcl-2: Proteintech, 12789-1-AP; Bax: Proteintech, 50599-2-Ig; Fas: Santa Cruz Biotechnology, sc-1023; FasL: Santa Cruz Biotechnology, sc-19681; β-actin: Santa Cruz Biotechnology, sc-130065), followed by incubation with horseradish peroxidase–conjugated secondary antibodies (Cell Signaling Technology, 1:1000) for 1 h at room temperature. Finally, PVDF membranes were treated with ECL Plus (Amersham Pharmacia Biotech, Umea, Sweden). Gray value of protein bands was analyzed using the ImageJ software (National Institutes of Health, U.S.A.). Expression of all target proteins was normalized to expression of β-actin.

Based on the previous study [21 (link)], 200 μL Radioimmunoprecipitation (RIPA) solution (Beyotime, Shanghai, China), which contains 1x protease inhibitor, was added to crack the crypts and shaken at 4 °C for 20 min to fully crack the crypts. The protein concentration was determined by Bicinchoninic Acid (BCA) kit (Beyotime, Shanghai, China). After adding the protein loading buffer, the protein was denatured at 100 °C for 10 min and stored at −80 °C refrigerator. The protein samples with the calculated concentration were subjected to Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) gel electrophoresis. Then, they were gummed, transferred, sealed, and incubated primary antibody overnight at 4 °C. Protein primary antibody information and dilution ratio: BAX (1:1000, Proteintech, Wuhan, China), Caspase9 (1:1000, CST, Boston, MA, USA), PCNA (1:1000, Abcam, Cambridge, UK), Olfm4 (1:1000, CST, Boston, MA, USA), P-YAP (1:1000, Abclonal, Wuhan, China), YAP (1:1000, Abcam, Cambridge, UK), Cyclin D1 (1:1000, CST, Boston, MA, USA). Finally, we incubated secondary antibodies, and the ECL solution was used to reveal the bands. A total of 30 experimental animals C57BL/6 mice were used.

The Day 28 was considered as an experimental endpoint. The tumors were weighed after mice were sacrificed. The removed tumors were used to prepare paraffin sections, which were then performed immunofluorescent (IF) staining for terminal deoxynucleotidyl transferase-mediated deoxy uridine triphosphate nick end labeling (TUNEL) (Roche, Germany) and immunohistochemical (IHC) staining of tumor apoptosis-related markers: p53 (1:400), Cyt C (1:400), Bcl-2 (1:400), which were obtained from Bioss (China) and Bax (1:100), which was purchased from Proteintech (USA). Quantification data were performed by calculating the mean optical density value of positive-staining areas versus the whole areas in five randomly selected fields using Image-Pro Plus software (Media Cybernetics).

Baicalein (≥ 99%, Yousi Scientific Co., Ltd, Shanghai, China) was dissolved in DMSO at concentration of 200 mM and stored at -20 ℃. Mdivi-1, an inhibitor of Drp1, was purchased from Selleck (Huston, TX, USA). 3-Methyladenine (3-MA), an inhibitor of autophagosomes, and Bafilomycin A1 (Baf-A1), an inhibitor of H⁺-ATPase, were purchased from Selleck. Antibodies against PARP (#9542), Drp1 (#5391), AMPKα (#5831), p-AMPKα (Thr172) (#2535), LC3 (#12741), Bak (#6947) and β-actin (#3700) were obtained from Cell Signaling Technology (Boston, MA, USA). Antibodies against Caspase 3 (#19677-1-AP), Caspase 9 (#10380-1-AP), Bcl2 (#12789-1-AP), Bcl-xl (#10783-1-AP), Bax (#50599-1-AP), Cytochrome c (#10993-1-AP), Aif (#17984-1-AP), Cox IV (#11242-1-AP), Fis1 (#10956-1-AP), Opa1 (#27733-1-AP), Mfn1 (#13798-1-AP), Ndufs1 (#12444-1-AP), Sdha (#14865-1-AP), Uqcrc1 (#21705-1-AP), Atp5a1 (#14676-1-AP), p62 (#18420-1-AP), and Beclin1 (#11306-1-AP) were obtained from Proteintech (Wuhan, China). Antibody against p-Drp1 (Ser616) (#12749) was obtained from Signalway Antibody (College Park, MD, USA). Secondary goat anti-rabbit or rabbit anti-mouse antibodies were purchased from Proteintech. Fluorescent-labeled antibody Annexin V-FITC, Annexin V-APC, PI, 7-AAD and 10 × binding buffer were obtained from BD (Franklin Lakes, New Jersey, USA).