Enhanced chemiluminescence reagent

Western blot was performed as described previously [17 (link)] using samples from differentiated HepaRG cells treated with T090 alone or in combination with SSM for 24 h. Total protein samples were obtained after RIPA buffer extraction. The buffer was supplemented with a phosphatase inhibitor cocktail for phosphorylated protein detection. Then, samples were subjected to electrophoresis in 10% SDS-PAGE gels. The separated proteins were then transferred to a nitrocellulose membrane (Amersham Biosciences, Buckinghamshire, UK). Membranes were incubated with the following primary antibodies: anti-SREBP-1c, ACLY, FAE, ACC, and S14 antibodies (purchased from Novus Biologicals, Centennial, CO, USA), FAS, SCD, SRC-1, and β-actin antibodies (purchased from Santa Cruz, Dallas, TX, USA), and SMILE and phosphoAMPK antibodies (purchased from GeneTex, Irvine, CA, USA). Then, the blots were probed with the appropriate secondary antibodies, and bands were visualized using an enhanced chemiluminescence reagent (Merck Millipore, Billerica, MA).

T24 and 5637 cells and tumor tissues were lysed using RIPA protein extraction reagent (Beyotime Institute of Biotechnology) supplemented with 1% protease inhibitor cocktails (Roche Applied Science). Protein concentration was measured using the BCA assay (Beyotime Institute of Biotechnology; cat. no. P0012). Equal amounts of proteins (20 µg/lane) were separated by 10% SDS-PAGE and transferred onto PVDF membranes (EMD Millipore). Following blocking with 5% non-fat milk at 4°C overnight, the membranes were probed with rabbit anti-HDAC2 monoclonal antibodies (1:2,000; Cell Signaling Technology, Inc.; cat. no. 57156s), mouse anti-β-actin monoclonal antibodies (1:3,000; Cell Signaling Technology, Inc.; cat. no. 3700), or rabbit anti-GAPDH (1:5,000; cat. no. sc-25778; Santa Cruz Biotechnology, Inc.) antibodies at room temperature for 1 h, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (1:4,000; OriGene Technologies, Inc.; cat. no. ZDR-5307, goat anti-mouse IgG/HRP and cat. no. ZDR-5306, goat anti-rabbit IgG/HRP) at room temperature for 1 h. Enhanced chemiluminescence reagent (Merck KGaA) was used to detect the signal on the membrane (Beijing Transgen Biotech Co., Ltd.).

Prepared cell lysates were separated by SDS-PAGE after boiled at 100°C for 5 min with 5 × SDS Loading buffer and transferred to Immobilon-P membrane (Merck Millipore). The membrane was blocked with 5% skimmed milk in Tris-buffer saline with 0.1% Tween 20. Then the membrane was incubated with indicated primary antibody, followed by a secondary antibody conjugated with horseradish peroxidase (HRP). Imaging was detected in LAS 4000 (Fujifilm) after incubating in an enhanced chemiluminescence reagent (Merck Millipore) for 1 min. The images’ brightness and contrast were adjusted using Multi Gauge software and the backgrounds were subtracted using ImageJ. To measure the integrated densities, the intensities of regions of interest were selected for all bands and normalized to that of corresponding Actin.

Western blotting was performed to measure the protein expression of p62, LC3-II, Beclin-1, NF-κB p65, Akt, p-Akt, mTOR, and p-mTOR. RAW264.7 cells were seeded into a six-well cell culture plate at a density of 40 × 10⁴ cells/well. After treatment, the cells were lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology) which contained protease inhibitors, phosphatase inhibitors, and PMSF at 4 °C for 30 min, and then, all soluble lysates were collected by centrifugation at 1.2 × 10⁴ rpm for 30 min at 4 °C. The lysates were boiled at 100 °C for 15 min to denature the proteins, and a BCA kit was used to measure the protein concentration. Protein samples (10 μL per lane) were separated on the basis of molecular mass by SDS–PAGE and then transferred onto a polyvinylidene difluoride membrane. The membranes were blocked with 5% skim milk powder for 1 h at room temperature and then incubated with primary antibodies for 24 h at 4 °C. Subsequently, the membranes were washed three times with Tris-buffered saline with 0.1% Tween-20, followed by incubation with a secondary antibody for 1 h at room temperature. After the membrane was washed three times, an enhanced chemiluminescence reagent (Merck KGaA) was added to the membrane, and the proteins which quantified with a gel imaging system (Bio-Rad Laboratories, Inc.).

The GSCs were lysed with RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing protease and phosphatase inhibitors for 30 min at 4 °C. Then the supernatants were collected after centrifugation at 12,000 rpm at 4 °C for 15 min. The supernatants were mixed with loading buffer (Boster, Wuhan, China), and equal amounts of protein were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, and then transferred to PVDF membranes. The PVDF membranes were blocked and incubated with the primary antibodies at 4 °C overnight. The primary antibodies were used at the following dilutions: rabbit anti-β-actin (1:2000; #4970, CST), mouse anti-β-III tubulin (1:1000; #4466, CST), rabbit anti-GFAP (1:1000; BA0056, Boster), mouse anti-sox2 (1:1000; ab171380, Abcam), rabbit anti-CD133 (1:1000; ab19898, Abcam), rabbit anti-H3 (1:1000; #4499, CST), rabbit anti-OCT4 (1:1000; ab18976, Abcam), rabbit anti-acetyl-H3 (1:1000; #06-599, Millipore), rabbit anti-H3K27me3 (1:1000; #9733, CST), mouse anti-H3K9me3 (1:1000; #5327, CST), rabbit anti-EZH2 (1:1000; #5246, CST). After washing with TBST, the membranes were incubated with HRP-conjugated goat anti-mouse or goat anti-rabbit secondary antibodies at room temperature. The antibody labeling was detected using an enhanced chemiluminescence reagent (Merck Millipore). The protein bands were analyzed using ImageJ.