Cleaved caspase 3

Total proteins were extracted from homogenized tissues and cell lysates using RIPA buffer containing protease inhibitors and phosphatase inhibitors (Pierce, Rockford, USA). An equal amount of protein lysates was separated on 12% SDS-PAGE and then transferred to polyvinylidene fluoride membranes. After an overnight incubation with the following primary antibodies at 4 °C: HOXB5 (1:1000, #109375, Abcam, Cambridge, UK), SESN3 (1:800, #97792, Abcam), IGF1R (0.1 μg/mL, #AF-305-SP, R&D Systems, Inc., Minneapolis, USA), protein kinase B (Akt, 1:1000, #9272, Cell Signaling), phosphorylated Akt (p-Akt, 1:1000, #9271, Cell Signaling, Danvers, USA), PCNA (PCNA, 1:1000, #18197, Abcam), p27 (1:1000, #32034, Abcam), B-cell lymphoma 2 (Bcl-2, 1:800, #59348, Abcam), E-cadherin (1:2000, #40772, Abcam), N-cadherin (1:1000, #76057, Abcam), cleaved caspase-3 (1:1000, #49822, Abcam), and GAPDH (1:2000, #37168, Abcam), the membranes were washed with Tris-Buffered Saline and stained with secondary antibody (1:2000, #6721, Abcam) for 60 min at room temperature. The images were captured using Alphalmager™ 2000 Imaging System (Alpha Innotech, USA).

The lower lobe of the left lung was homogenized in lysis buffer, and the proteins were collected. For the detection of Nrf2 and NF-κB, the cytoplasmic component and nuclear component were isolated by treating a nuclear protein extraction kit (Beyotime Biotechnology, Shanghai, China) and centrifuged at 12,000 g for 10 min at 4°C. After the protein concentration was measured by a BCA kit, 50-μg protein samples were separated by SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked in 5% non-fat dry milk, followed by incubation with pro-caspase-3 (1:8,000, Abcam, United States), cleaved-caspase-3 (1:500, Abcam, United States), pro-caspase-9 (1:800, Abcam, United States), cleaved-caspase-9 (1:1,000, Abcam, United States), Nrf2 (1:5,000, Abcam, United States), and NF-κB (1:3,000, Abcam, United States) primary antibodies overnight at 4°C. After being washed and incubated with secondary antibody (1:5,000, Zhongshan Golden Bridge Biotechnology, Beijing, China) at room temperature for 1 h, the proteins were visualized with the enhanced chemiluminescence reagent (GE Healthcare Bio-Sciences, Pittsburgh, PA, United States), analyzed using ImageJ version 1.61 software (National Institutes of Health, Bethesda, MD, United States) and normalized to β-actin and Lamin B.

NSCLC cells were lysed in RIPA lysis buffer (KeyGEN, Nanjing, China), and the concentration of protein was detected by BCA Assay kit (Solar life science, Beijing, China). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 10%) was performed to separate the equal amounts of protein (30 μg), and proteins were then shifted onto polyvinylidene difluoride membrane (PVDF, Thermo Fisher Scientific). Five percent nonfat dried milk in TBST was applied to block the PVDF membrane for 1 h. PVDF membrane was incubated at 4°C overnight with the primary antibodies: E-cadherin (Abcam, Cambridge, MA, USA, 1:1000), N-cadherin (Abcam, 1:1000), p-Akt (Abcam, 1:1000), Akt (Abcam, 1:1,000), p-ERK (Abcam, 1:1000), ERK (Abcam, 1:1000), cleaved caspase 3 (Abcam, 1:1000), p27 Kip1 (Abcam, 1:1000), cyclin D1 (Abcam, 1:1000), CDK2 (Abcam, 1:1000) and β-actin (Abcam, 1:1000). Then, the membranes were incubated with HRP-conjugated secondary antibodies (Abcam; 1:5000) for 1 h. Protein bands were visualized using the ECL kit (Thermo Fisher Scientific). β-actin was used as the loading control. IPP 6.0 (Image-Pro Plus 6.0) was used for the densitometry analysis.

DHM (purity ≥99.0%) and TAA (purity ≥99.0%) were purchased from Sigma. Hematoxylin-eosin (H and E), TUNEL and Masson stain kits, and commercial assay kits for ALT and AST, SOD, GSH, MDA were obtained from the Nanjing Jiancheng Bioengineering Research Institute (Nanjing, China). The immunofluorescent staining kits and the enhanced chemiluminescence (ECL) kit were purchased from Beyotime science and technology Co., Ltd. (Beijing, China). The antibody of rabbit monoclonal α-SMA, TGF-β1, CYP2E1, Cleaved Caspase-3, Caspase3, Cleaved Caspase-9, Caspase-9, IκB kinase α (IKK-α), IκB kinase β (IKK-β), p-IKKα, p-IKKβ, inhibitor of IκBα (IκBα), p-IκBα, NF-κB, p-NF-κB, PI3K, p-PI3K, Akt, p-Akt, B-associated X (Bax), Bcl-2, β-actin and secondary antibodies for western blot were all obtained from Abcam (Cambridge, United Kingdom).

After washing twice with ice-cold phosphate-buffered saline (PBS), the cells were incubated at 4 °C with RIPA buffer supplemented with a proteinase inhibitor cocktail (JRDUN biotech, Shanghai, China) for 30 min. Subsequently, the samples were centrifuged at 12,000 r.p.m. for 15 min at 4 °C, and the supernatant was collected for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein on the gels were electrophoretically transferred to a nitrocellulose membrane (Millipore, Bredford, MA, USA), and probed with primary antibodies against TRIM65 (Abcam, Cambridge, MA, USA; 1:1000 dilution), TNRC6A (Abcam; 1:1000 dilution), ATG7 (Abcam; 1:200 dilution), LC3 (Abcam; 1:2000 dilution) or cleaved caspase3 (Abcam; 1:1000 dilution). After incubation with an HRP-conjugated secondary antibody (Beyotime, Shanghai, China; 1:1000 dilution), signals were detected using an enhanced chemiluminescence (ECL) detection kit (Millipore). The membrane was probed with a primary antibody against GAPDH (Cell Signaling Technology, Danvers, MA, USA; 1:2000 dilution) for equal loading control.

Total proteins were extracted from B-CPAP cells using radioimmunoprecipitation assay and quantified using a bicinchoninic acid assay kit (Solarbio). In brief, 20 μg of harvested proteins from each group was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were then blocked with 5% skim milk at 4°C overnight and cultured for 1 h with primary antibodies against B cell lymphoma-2 (Bcl-2), Bcl-2/Bcl-XL-associated death promoter (Bad), lactate dehydrogenase A (LDHA), hexokinase 2 (HK2), Akt (all supplied by Bioswamp), cleaved caspase-3, phosphorylated (p)-Akt, and GAPDH (an endogenous control) (supplied by Abcam). Subsequently, the membranes were incubated with goat anti-rabbit IgG secondary antibody for 1 h.