Protease inhibitor

The liver tissues were collected for protein extraction as described previously (Liu et al., 2021 (link)). In brief, total protein was extracted from the liver tissue using RIPA protein extraction solution (Boster Biological Technology, China) supplemented with protease inhibitors (Boster Biological Technology, China). The supernatant was centrifuged at 12,000 × g for 15 min. The protein concentration was measured and adjusted by BCA Protein Assay kit (Beyotime Biotechnology, China). Equal amounts of protein samples (10 μg) from the liver homogenates were separated by 10% SDS‐PAGE and transferred to a polyvinylidene fluoride membranes (Millipore, USA). After blocking with 5% skimmed milk for 1 h, the membrane was incubated overnight with the flowing antibodies at 4°C including anti‐CD36 (1:1000), anti‐HMGCS2 (1:1000), anti‐ACSL1 (1:1000), anti‐FADS2 (1:500), anti‐ACAA1 (1:500), anti‐CYP7A1 (1:500), and anti‐GAPDH (1:8000). After washing with TBST for five times, these membranes were incubated with goat anti‐rabbit horseradish peroxidase for 1 h at room temperature. Positive bands were detected by enhanced chemiluminescence detection system (Bio‐Rad Laboratories, USA). Image J software was used to quantify the immunoblots.

Subsequent to trypsin treatment, cells were lysed with enhanced radio-immunoprecipitation assay (RIPA) lysis encompassing protease inhibitors (BOSTER, Wuhan, Hubei, China), followed by estimation of protein concentration using Bicinchoninic Acid (BCA) Protein Quantification Kit (BOSTER). Proteins underwent separation by 10% sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE). Then, the separated proteins were electroblotted into a polyvinylidene fluoride (PVDF) membrane which was sealed by 5% bovine serum albumin to block nonspecific binding. Overnight cell incubation was conducted at 4 °C after supplementation with primary rabbit antibodies (Abcam, Cambridge, UK) to Cleaved caspase-3 (ab49822, 1:500), Bcl-2-Associated X (Bax, ab32503, 1:1000), B-cell lymphoma-2 (Bcl-2, ab196495, 1:500), MDM2 (ab226939, 1:3000), PPARγ (ab45036, 1:500), Ubiquitin (ab7780, 1:2000), and β-actin (ab8227, 1:500). Then, horseradish peroxidase-tagged goat anti-rabbit secondary antibodies (ab205719, 1:2000, Abcam) were supplemented for 1-h membrane incubation at 4 °C. After development in ECL working fluid (EMD Millipore Corporation, Billerica, MA, USA), the bands in Western blot images were quantified by Image J analysis software by normalizing to β-actin.

Tumor tissues and cells were washed with PBS and underwent lysis with RIPA buffer (Pierce) supplemented with protease inhibitors (Boster Biological Technology, China). Protein quantitation utilized the BCA assay kit (Beyotime Biotechnology). Equal amounts of total protein were resolved by SDS-PAGE and underwent transfer onto polyvinylidene fluoride membranes (Millipore, USA). Western blot membranes underwent overnight incubation at 4 °C with anti-FAM126A (1:1000; Sigma, #84668), anti-ENO1 (1:1000; Proteintech, #11204), anti-GHPDH (1:1000; Proteintech, #60004), anti‑E‑cadherin (1:1000; Proteintech, #20874), anti-N-cadherin (1:1000; Proteintech, #22018), anti‑vimentin (1:1,000; Proteintech, #10366), anti-snail (1:1000; Proteintech, #13099), anti-cyclin D1 (1:1000; Cell Signaling Technology [CST], #55506), anti-cyclin E1 (1:1000; CST, #4136), anti-CDK 2 (1:1000; CST, #2561), anti-CDK4 (1:1,000; CST, #12790), anti-AKT (1:1000; CST, #4691), anti-p-AKT (1:2000; CST, #4060), anti-PI3K (1:1000; CST, #4249), anti-p-PI3K (1:1000; CST, #17366), anti-Ki-67(1:800; CST, #9449) and anti-PCNA (1:1000; CST, #2561) primary antibodies. This was followed by incubation with HRP-linked anti-rabbit IgG (Boster, #BA1041). Enhanced chemiluminescence reagent (Proteintech, #7003) was used to detect immunoreactive signals.

For the analysis of Bcl2, bax, LC3B, osteosarcoma cells were resuspended in a protein lysis buffer RIPA (Boster bio, Wuhan, China) containing protease inhibitors (Boster Bio, Wuhan, China) and phosphatase inhibitor (Boster Bio, Wuhan, China) at 4°C for 30 min incubation. After homogenization, the supernatants containing protein was collected by centrifugation and stored at –80°C. Followed by the BCA protein assay, protein samples were separated by 10%–12% SDS-PAGE electrophoretically and were then transferred to a PVDF membrane (GE Healthcare life Sciences, Piscataway, USA). Then western blotting were proceeded with primary and secondary antibodies and was detected with peroxidase-conjugated anti-rabbit antibody followed by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, USA). The details of the primary antibodies and dilution factors were as follows: Bcl2 (Cell Signaling Technology, Inc. USA, 1:1000), bax (Cell Signaling Technology, Inc. USA, 1:1000), LC3B (Cell Signaling Technology, Inc. USA, 1:1000), GAPDH (Cell Signaling Technology, Inc. USA, 1:1500).

Oroxin B (OB, HY-N1435, purity: 99.71%, CAS number: 114482-86-9), Dimethyl sulfoxide (DMSO, HY-Y0320, purity: ≥99.0%, CAS number: 67-68-5), and autophagy inhibitor 3-methyladenine (3-MA, HY-19312, purity: 99.83%, CAS number: 5142-23-4) were acquired from MedChemExpress (Monmouth Junction, NJ, USA). Recombinant mouse IL-1β was purchased from R&D systems (Minneapolis, USA). Boster (Wuhan, Hubei, China) provided the RIPA lysis buffer, protease inhibitors, phosphatase inhibitors, BCA protein assay kit, protein loading buffer, rabbit/mouse secondary antibodies, and DAPI reagent. The ECL chemiluminescent substrate was obtained from Bio-Rad (Hercules, CA, USA). The biotechnology of Biosharp Life Sciences (Hefei, Anhui, China) provided the Trypsin (BL527A) and collagenase II (BS164). DMEM/F12 medium culture was got from Hyclone (Logan, UT, USA). Foetal bovine serum (FBS) was purchased from BioInd (Biological Industries, Israel). Omega Bio-tek (USA) provided the RNA extraction kit (R6834-01). Yeasen (Shanghai, China) provided the kits for complementary DNA (cDNA) synthesis (11141ES60) and quantitative real-time polymerase chain reaction (RT-qPCR) (11201es08).

Total cell protein was extracted on ice with RIPA lysis buffer (Beyotime biotechnology, Shanghai, China) in the presence of freshly added protease inhibitors (Boster Biological Technology, Wuhan, China), and quantified by the BCA assay (Pierce, Cramlington, UK). A total of 30 μg protein from each sample was subjected to 10% SDS–PAGE (Beyotime biotechnology, Shanghai, China) by electrophoresis under reducing conditions and transferred to PVDF membrane (Millipore Corporation, Billerica, MA, USA). The blotted membrane was then blocked with 5% skim milk for 1 h at room temperature and incubated respectively with anti-ETAR (1:5000, ab117521, Abcam, Cambridge, MA), ETBR (1:5000, ab117529, Abcam, Cambridge, MA) and anti-GAPDH (1:2000, 10494-1-AP, ProteinTech, Chicago, USA) antibody overnight at 4°C. The membranes were further incubated with HRP-conjugated anti-rabbit secondary antibodies and detected using the enhanced chemiluminescence (ECL; Abbkine, Redlands, CA, USA) method. The densitometry was performed using Image J software.

Total protein was extracted from cells using cell lysis buffer (Boster) containing protease inhibitors (Boster, Wuhan, China). Total proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis on 10% gels, and the separated proteins were transferred to polyvinylidene fluoride membranes (Millipore, NY, USA). After blocking with 3% BSA for 2 h, the membranes were incubated with primary antibodies against the following proteins: YAP (1 : 500, Boster, Wuhan, China), COL I (1 : 1000; Santa Cruz, USA), osteocalcin (OCN; 1 : 1000; Abcam, UK), RUNX-2 (1 : 1000; Abcam, UK), osteopontin (OPN; 1 : 1000; Boster), TG2 (1 : 1000; Santa Cruz, USA), HSP70 (1 : 500, Bioss, Beijing, China), and YAP (1 : 500, Boster, Wuhan, China). HRP-conjugated goat anti-rabbit IgG (1 : 5000; Boster) was applied as a secondary antibody and incubated with the membrane for 1 h at room temperature. Immunoreactive bands were detected using enhanced chemiluminescence reagents (Millipore, NY, USA). Signal intensity was measured using a Bio-Rad XRS chemiluminescence detection system (Bio-Rad, CA, USA). Actin or β-tubulin served as the loading control.