P akt

Proteins within tumour tissues and HUVEC cells were extracted using a Boster Kit (Bosterbio, CA, USA) according to the manufacturer’s instructions. Western blotting was performed as previously described [20 (link)]. The antibody, which was raised against the Cyp2c44’s IGRHQPPSMKDKMKC peptide (GenScript), was generated according to previous studies [13 (link), 16 (link)]. Other antibodies used in this study are as follows: Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Bosterbio), β-actin (Bosterbio), Cyp2c9 (Abcam, Cambridge, UK), COX1 (Santa Cruz, CA, USA), COX2 (Santa Cruz), Phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) (Santa Cruz), ERK1/2 (Santa Cruz), P-AKT (Abcam), AKT (Abcam), and CD31 (Abcam).
For qRT-PCR, RNA from tumours was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and reverse-transcribed using the M-MLV First-Strand cDNA Synthesis Kit (Invitrogen) [15 (link)]. The mRNA levels of target genes were quantified by qRT-PCR using Power SYBR Green PCR Master Mix (Invitrogen) with the primers listed in Additional file 1: Table S1. GAPDH served as an internal control and the results were analysed using the 2^-ΔΔCt method.

In this study, proteins were extracted using a protein extraction kit (Sangon Biotech), and the bicinchoninic acid (BCA) assay (Sangon Biotech) was used to determine the total protein content. After denaturation for 5 min, total proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA) via a constant current flow at 200 mA. Subsequently, the PVDF membranes were incubated with Bcl-2 (1:10,000), Bax (1:2000), Caspase-3 (1:5000), M-CSF (1:1000), RANKL (1:3000), OPG (1:3000), p-p65 (1:1000), p65 (1:1000), p-p38 (1:1000), p38 (1:5000), p-JNK (1:1000), JNK (1:1000), p-AKT (1:2000), AKT (1:2000), p-ERK (1:1000), ERK (1:1000), RANK (1:2000), NF-κB (1:1000), NFATc1 (1:10,000), and c-Fos (1:1000) antibodies (Abcam, Cambridge, MA) for 12 h at 4°C. TBS buffer was used to wash the PVDF membranes, and secondary antibodies (Abcam) were added and incubated at room temperature for 1 h. After the membranes were washed three times, chemiluminescent reagents were added, and the grayscale values of the bands were analyzed using ImageJ software. Each experiment was independently repeated 3 times. GAPDH was used to quantify the expression of various proteins.

Total protein was extracted from lung tissue homogenates using the RIPA reagent (Beyotime Biotechnology, lnc, JiangSu, China) supplemented with 1% PMSF (Beyotime) and separated through electrophoresing on 10% SDS-PAGE gels (30μg/lane). The separated protein was transferred to the polyvinylidene difluoride (PVDF) membranes (Merck Millipore, lnc., Darmstadt, Germany) and subsequently blocked with 5% skimmed milk. The targeted protein was probed with antibodies against INSC, AKT, p-AKT, mTOR, p-mTOR, and β-actin (all from Abcam, Cambridge, United Kingdom). After an incubation step with horseradish peroxidase (HRP)-conjugated secondary antibodies, the bands were visualized using an enhanced chemiluminescence kit (Merck Millipore).

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).

Cells were lysed in 100 μl RIPA buffer (MedChemExpress). Proteins were transferred from SDS‒PAGE gels to Immobilon-FLPVDF membranes, blocked, and then incubated with primary antibodies overnight at a 1:1000 dilution. Antibodies against CCR5, AKT, p-AKT, Sp1, CD44, EpCAM, c-Myc, LATS 1, YAP, and GTGF (Abcam) were used. Membranes were then incubated in secondary antibodies, washed, and exposed on a chemiluminescence imaging system (Beijing Sage Creation Science) with ECL (Thermo Fisher Scientific). Western blotting was repeated three times for each band (see Additional file 1).