Wl01863

Paraffin-embedded sections (4 μm thick) were used for IHC. After deparaffinisation, hydration, and antigen retrieval at RT, sections were incubated with antibodies specific for FTO (27226-1-AP, Proteintech, 1:500), SNAIL (WL01863, Wanleibio, Shenyang, China, 1:500), or Ki-67 (D2H10, Cell Signalling Technology, Boston, MA, USA, 1:800) at 4 °C overnight. On the second day, the sections were processed as described previously [35 (link)]. Two independent pathologists scored the specimens according to the percentage of positively stained cells (0 = 0–4%; 1 = 5–25%; 2 = 26–50%; 3 = 51–75%; 4 = 76–100%) and the intensity of staining (0 = none, 1 = slight, 2 = moderate, 3 = strong). The percentage and intensity were multiplied to obtain the total score of one visual field. The IHC score was equal to the average of the total scores for each visual field.

Total protein was extracted using a cell lysis buffer for Western blotting and immunoprecipitation (Beyotime, Shanghai, China) containing PMSF and a protease inhibitor cocktail. Proteins (30 μg/well) were separated using 10% SDS/PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The primary antibodies used in this study were against anti-HAPSTR1 (1:1000, OTl2B8; Origene), anti-HAPSTR1 (1:500, OTI2D1; Novusbio, Centennial, CO, USA), anti-LRPPRC (1:5000, 21175-1-AP; Proteintech), anti-β-actin (1:1000, 20536-1-AP; Proteintech), anti-Ub (1:1000, 10201-2-AP; Proteintech), anti-N-cadherin (1:5000, 66219-1-Ig; Proteintech), anti-Beclin1 (1:2000, T55092; Abmart, Berkeley Heights, NJ, USA), anti-P62/SQSTM1 (1:2000, T55546; Abmart), anti-ATG5 (1:2000, T55766; Abmart), anti-vimentin (1:500, WL01960; Wanleibio), anti-snail (1:500, WL01863; Wanleibio), and anti-E-Cadherin (1:500, WL01482; Wanleibio).

Radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, Shanghai, China) was used for protein extraction. Proteins were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to a 0.22 μm PVDF membrane. The membrane was blocked with 5% non-fat milk and subsequently incubated with primary antibodies at 4 °C overnight. The primary antibodies used were as follows: GAPDH (10494-1-AP, Proteintech, Wuhan, China, 1:10,000), FTO (27226-1-AP, Proteintech, 1:3000), E-cadherin (20874-1-AP, Proteintech, 1:5000), N-cadherin (22018-1-AP; Proteintech, 1:3000), vimentin (60330-1-Ig, Proteintech, 1:10,000), ZEB1 (21544-1-AP, Proteintech, 1:3000), ZEB2 (14026-1-AP, Proteintech, 1:500), SNAIL (WL01863, Wanleibio, Shenyang, China, 1:500), SLUG (WL01508, Wanleibio, 1:500), and IGF2BP2 (11601-1-AP, Proteintech, 1:4000). The next day, the membrane was visualised using an ECL detection system after incubation with their respective secondary antibody.