Af6242

Total protein was collected using RIPA buffer containing a mix of protease inhibitors and quantified with a BCA protein assay kit. Cell lysates (30 μg or 50 μg per sample) were resolved by SDS-PAGE, and then the proteins were transferred to PVDF membranes. After being incubated in blocking buffer, the PVDF membranes were incubated with primary antibodies against PI3K (1:1000, Affinity, AF6242), p-PI3K (1:1000, Affinity, AF3242), AKT (1:1000, Affinity, AF6261), p-AKT (1:1000, Affinity, AF0016), GAPDH (1:7500, Proteintech), Snail (1:1000, CST), vimentin (1:1000, CST), β-catenin (1:1000, CST), E-cadherin (1:1000, CST) and PAI-1 (1:1000, CST) at 4 °C overnight. A secondary goat anti-rabbit HRP-IgG (Proteintech) was used to detect the primary antibodies, and the chemiluminescence intensities were detected using the ECL system (Millipore, Billerica, USA).

Deparaffined and hydrated liver tissue sections were immunostained for PTEN and PI3K using rabbit anti-human antibody against PTEN (ab170941, 1:200, Abcam, Cambridge, UK) and PI3K (AF6242, 1:100, Affinity, Jiangsu, China). Visualization was performed using 3,3-diaminobezidine (DAB; ST033, Whiga Biosmart Co., Ltd., Guangzhou, China). Five high-power fields of view were randomly captured for each replicate and 100 cells in total were counted in each field using a microscope. Staining intensities were scored as 0 and 1 (negative), 2 (moderate) and 3 (positive). Ten fields were randomly selected at 200 × magnification and observed. The staining intensities were divided into four grades (0–3 points): 0 indicates invisible positive staining; 1 indicates faint staining; 2 indicates moderate staining; 3 indicates strong positive staining. The proportion of positive cells was also scored from 0 to 3 points, in which 0 indicates tumor cells without positive staining, 1 indicates positive cells less than 20%, 2 indicates 20–50% of positive cells, and 3 indicates more than 50% positive cells. Final staining scores, consisting of intensity and proportion scores, 0–1 were defined as negative/weak, 2–4 as positive and 6–9 as strong positive.

Cells were dealt with as cell cycle and apoptosis analysis described above. Whole‐cell extracts were prepared, and then separated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) before being transferred to nitrocellulose membranes (Millipore, USA). Antibodies used in this study included cyclin‐dependent kinase 1 and 2 (CDK1 and CDK2; DF6024 and AF6237, Affinity, USA), cyclinA2 (18202‐AP, Proteintech, China), cyclinB1 (28603‐1‐AP, Proteintech, China), B‐cell lymphoma‐2 (Bcl‐2; 12789‐1‐AP, Proteintech, China), Bcl‐2 associated X (Bax; 50599‐2‐Ig, Proteintech, China), vimentin (ab128507, Abcam, UK), E‐cadherin (20874‐1‐AP, Proteintech, China), N‐cadherin (22018‐1‐AP, Proteintech, China), ICAM2 (DF6772, Affinity, USA), PI3K (AF6242, Affinity, USA), p‐PI3K (AF3242, Affinity, USA), AKT (4691, CST, USA), p‐AKT (4060, CST, USA), p300 (AF5360, Affinity, USA), METTL3 (ab195352, Abcam, UK) and β‐actin (4970, CST, USA), as well as anti‐rabbit IgG horseradish peroxidase‐linked antibody (7074, CST, USA). ChemiDoc XRS+ (Bio‐Rad, USA) was adopted for capturing images.