Cell lysate

Sprague-Dawley (SD) rats were purchased from Vital River Laboratories Animals Technology Co., Ltd. (Beijing, China). PIO was donated by Huadong Medicine Co., Ltd. (Hangzhou, China). GW9662, diphenylene iodonium (DPI) and pyrrolidine dithiocarbamate (PDTC) were purchased from Sigma (St. Louis, MO, USA). Dulbecco’s modified Eagle medium (DMEM) and fetal bovine serum (FBS) were purchased from Hyclone Laboratories, Inc. (Logan, UT, USA). TRIzol^® reagent and cell lysates were purchased from Invitrogen Life Technologies (Shanghai, China). Anti-GAPDH was purchased from ProMab Biotechnologies Inc. (Richmond, CA, USA) Anti-RAGE was obtained from Abcam (Hong Kong, SAR, P.R. China). Anti-NF-κB p65, anti-I-κBα, fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG and the reactive oxygen species (ROS) assay kit were purchased from the Beyotime Institute of Biotechnology (Jiangsu, China).

MiR-524-5p no-biotin probe and miR-524-5p biotin probe were synthesized by Thermo Fisher Scientific. The biotinylated miRNA was incubated with cell lysates (Invitrogen) overnight, followed by adding streptavidin magnetic beads (Invitrogen). Finally, RT-qPCR was used to detect the expression levels.

The treated cells were obtained and resuspended in cell lysate (Thermo) on ice for at least 30 min. The cell lysate was centrifuged at 12 000 × g for 10 min at 4°C. The supernatant was obtained, mixed with 5 × loading buffer, and boiled for 10 min. After boiling, the resulting supernatant containing total protein was quantified using the BCA protein evaluation kit (Beyotime P0012). An 8%, 10%, or 15% polyacrylamide gel (depending on the protein's molecular size to be analyzed) was used to separate a 50 µg total protein sample. The separated sample was transferred to a PVDF membrane (Millipore). The membrane was blocked using 5% skimmed milk and incubated with primary antibodies (1: 1000 in TBST) of cleaved caspase 3, cleaved caspase 8,Bax, Bcl‐2, P‐AMPK, P‐mTOR, p‐p70s6k, and GAPDH. Secondary antibodies coupled with horseradish peroxidase were then introduced into the membrane. The membrane was then incubated. Enhanced chemiluminescence (ECL) reagent (Pierce, Thermo Fisher Scientific) was used to detect protein expression.

Cells were collected and total protein were extracted using cell lysate (Thermo, USA), and the concentration of proteins was evaluated by a BCA kit (Beyotime, China). The proteins (20 μg) were separated by 10% SDS-PAGE and then transferred to the polyvinylidene difluoride (PVDF) membranes (Millipore, MA). After blocking with 5% skimmed milk for 1 h at room temperature, the membranes were incubated with the primary antibodies overnight at 4°C. Then it was rinsed three times and incubated with an HRP-conjugated secondary antibody at room temperature for 1 h. After washing the membrane three more times, we applied a chemiluminescence reagent for developing the proteins, which were placed under the gel imaging system for the image collection, and the gray value of the protein band was analyzed by ImageJ software for the calculation of the protein expression. The primary antibodies used in the study were as follows: E-cadherin (#14472, CST, USA), Vimentin (#5741, CST), N-cadherin (#13116, CST), Twist (ab50887, Abcam, UK), p-PI3K (ab278545, Abcam), PI3K (ab191606, Abcam), p-AKT (ab38449, Abcam), AKT (ab38449, Abcam), and GAPDH (ab8245, Abcam). The internal control was GAPDH.

Total RNA was extracted from cells grown onto 12-well transwells using TRI reagent (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions with some modifications. RNA was quantified using a Nanodrop Spectrophotometer (ND-1000, Labtech International, Uckfield, UK). RNA integrity was assessed by agarose gel electrophoresis. For cytoplasmic RNA extraction, 0.1% NP-40 was added to 100 μl cell lysate (Thermo Fisher Scientific). The cytoplasmic fraction was collected by centrifuging for 1 min at 14,000 rpm at 4°C. RNA purity and quantity were determined by Nanodrop Spectrophotometer. RNA integrity was assessed by agarose gel electrophoresis. All extractions were repeated twice for reproducibility. Complementary DNA (cDNA) was synthesized from 500 ng of total RNA from each sample using Superscript III First-Stand Synthesis System (Invitrogen).