Enhanced chemiluminescence

Total protein was extracted from HUVECs using a radio-immunoprecipitation assay lysis buffer (RIPA) with protease and phosphatase inhibitors (Cwbio, Beijing, China). 30μL protein lysates were separated by 10% sodium dodecyl sulfate polyacrylamide gel (Vazyme) electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore). Then, the membranes were blocked with 5% non-fat milk for 1 h at room temperature. Subsequently, they were incubated overnight at 4 °C with the following primary antibodies: monoclonal Mouse anti-METTL3 (1:1000, Santa Cruze), polyclonal rabbit anti-PGDF-BB (1:1000, Abcam), polyclonal rabbit anti-GAPDH or polyclonal rabbit anti-β-ACTIN (1:1000, Cell Signal Technology). After washing with tris-buffered saline and tween 20 (TBST), the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology) for 1 h at room temperature. After repeating the washing step, the target protein signal was measured by enhanced chemiluminescence (Thermo Scientific). Then, the membranes were visualized using a gel imaging system (Bio-Rad, Hercules, CA). The expression of protein was quantified by ImageJ software.

Cells were lysed in ice-cold RIPA buffer (1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 10 mM Tris pH 8 and 140 mM NaCl) supplemented with 250 μnits per ml Benzonase, 1 mM dithiothreitol and phosSTOP phosphatase inhibitor cocktail and a cOmplete protease inhibitor cocktail (both from Roche, Indianapolis, IN, USA). All other reagents were obtained from Sigma. Proteins were quantified using a BCA protein assay (Thermo Scientific, Waltham, MA, USA) and proteins (20 μg per sample) were separated using SDS–polyacrylamide gel electrophoresis and electroblotted to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA). Membranes were stained with Ponceau Red to monitor the transfer of proteins and then blocked with 5% non-fat dry milk (Bio-Rad). Membranes were probed with anti-Grp94 (Enzo Life Sciences, Farmingdale, NY, USA), anti-grp78, anti-CHOP/Gadd153, anti-phospho-Akt (T308, T450 and S473), anti-pan Akt, anti-phospho-GSK3β (S9), anti-GSK3β, anti-phospho-IRS-1 (Y632), anti-IRS-1, anti-survivin, anti-phospho-PP1α (T380), anti-PP1α (all obtained from Cell Signaling Technology, Danvers, MA, USA), anti-α-tubulin (Abcam, Eugene, OR, USA) and anti-β-actin (Chemicon, Temecula, CA, USA) antibodies. Following incubation with primary antibodies, membranes were washed, reacted with horseradish peroxidase–conjugated secondary antibodies and visualized using enhanced chemiluminescence (Thermo Scientific). For some experiments, band intensities were quantified using Image J (NIH, Bethesda, MD, USA).

Following treatment, the C17.2 cells were collected and lysed for protein expression analysis. The protein concentration was determined using the BCA protein assay kit (Thermo Fisher Scientific) with bovine serum albumin as a standard. Total protein equivalents were denatured and separated on a 10–15% sodium dodecyl sulfate (SDS)-polyacrylamide gel, and the proteins were then transferred onto Immobilon-P Transfer membranes (Millipore). The membranes were blocked with 5% non-fat dry milk for 1 h at room temperature and then incubated with primary antibodies. The primary antibodies were as follows: Hsp75 (ab151239; Abcam, Cambridge, MA, USA), CypD (A3208; ABclonal, Cambridge, MA, USA), Cytc (ab133504; Abcam), caspase-3 (WL0146; Wanleibio, Shenyang, China), COX IV (internal control for mitochondrial protein; ab140643; Abcam), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; KC-5G5) and β-actin (KC-5A08; both from KangChen Bio-tech, Shanghai, China) and α-tubulin (AT819; Beyotime Institute of Biotechnology, Haimen, China). The secondary antibodies were either goat anti-rabbit (BA1054; Boster Biotech Co., Ltd., Wuhan, China) or mouse (bs-0296G-HRP; Bioss Co., Beijing, China) immunoglobulin G (IgG). Horseradish-conjugated secondary antibody labeling was detected using enhanced chemiluminescence (Thermo Fisher Scientific) according to the manufacturer's instructions. The optical density of the bands was analyzed with ImageJ software.
For RT-qPCR, total RNA was extracted from the C17.2 cells using TRIzol^® reagent (Invitrogen, Carlsbad, CA, USA). The RNA (2 µg) was reverse transcribed into cDNA using ReverTra Ace^® qPCR RT Master Mix with the gDNA Remover kit (Toyobo, Osaka, Japan). Real-time monitoring of the PCR amplification of cDNA was detected using a real-time PCR detection system (ABI PRISM^® 7500 sequence detection system). PCR amplification was conducted as follows: 40 cycles at 95°C for 15 sec and an extension at 60°C for 32 sec. The data were quantified using the 2^−ΔΔCt method. The following primers were used: mouse CypD, 5′-AACTTCAGAGCCCTATGCA-3′ (forward) and 5′-TCCTGTGCCATTGTGGTT-3′ (reverse); mouse caspase-3, 5′-GTTCATCCAGTCCCTTTGC-3′ (forward) and 5′-TGTTAACGCGAGTGAGAATG-3′ (reverse); mouse β-actin, 5′-GCTTCTAGGCGGACTGTTAC-3′ (forward) and 5′-CCATGCCAATGTTGTCTCTT-3′ (reverse). β-actin (a housekeeping gene) was used as an internal reference.