Mettl16

Reverse transcription-quantitative PCR (RT-qPCR) and western blot methods were described previously 20 . The oligos and primers used in this study were listed in table S1. Moreover, the primary antibodies used in this study were as follows: GAPDH (cat. no. 2118, 1:1,000; Cell Signaling Technology), histone H3 (cat. no. 4499t, 1/1,000; Cell Signaling Technology), METTL16 (cat. no. A15894, 1/1,000; ABclonal), E-cadherin (cat. no. 20874-1-AP, 1/10,000; Proteintech), N-cadherin (cat. no. 66219-1-Ig, 1/5,000; Proteintech), ZO-1 (cat. no. 21773-1-AP, 1/5,000; Proteintech), DVL2 (cat. no. 12037-1-AP, 1/5,000; Proteintech), β-catenin (cat. no. A19657, 1/1,000; ABclonal), Phospho-β-catenin-S45 (cat. no. AP0580, 1/1,000; ABclonal), METTL3 (cat. no. A8370, 1/1,000; ABclonal), and METTL14 (cat. no. 26158-1-AP, 1/1,000; Proteintech).

The culture medium was discarded and SW480 cells were washed with PBS three times. Lysis buffer (1000 µL) was added into each dish, which were then shaken for 20 min. The cells at the bottom of the dish were scraped off using a brush and placed into the EP tube. The collected cells were lysed using an ultrasonic pyrolyser for about 15 s. After standing for 15 min, the cells were centrifuged at 12000 rpm for 0.5 h. The supernatant was separated into EP tubes, and the protein concentration was determined by ultraviolet spectroscopy. All protein samples were adjusted to the same concentration (1 mg/ml). Protein samples were sub-packaged and placed in the refrigerator at -80°C. The total protein from SW480 cells was then analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The protein in the gel was transferred onto a polyvinylidene difluoride (PVDF) membrane, incubated with the primary antibody METTL16 (1:1000, #17676 from Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight, and then incubated with a goat anti-rabbit secondary antibody (1:2000, #7074 from Cell Signaling Technology, Danvers, MA, USA) in a dark place for 1 h. The protein bands were scanned and quantified using an Odyssey scanner (LI-COR Biosciences, Lincoln, NE, USA), and the level of detected proteins was adjusted to GAPDH level.

Total protein extracts were isolated with Radioimmunoprecipitation assay (RIPA) buffer in the presence of protease and phosphatase inhibitor cocktail (Roche). Equal quantities of protein were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes, immunoblotted with the appropriate antibodies, and revealed using Odyssey. The antibodies used in this study were as follows: METTL16 (Cell Signaling Technology, #87,538), PRDM15 (Proteintech, 25,590–1-AP), SETD5 (Abclonal, A7304, RRID:AB_2767845), KMT2D (Proteintech, 27,266–1-AP), NSD2 (Abclonal, A7938, RRID:AB_2772896), FGFR4 (Proteintech, 67,800–1-Ig), YTHDF1 (Proteintech, 17,479–1-AP, RRID:AB_2217473), YTHDF3 (Cell Signaling Technology, #24,206), FLAG (Cell Signaling Technology, #14,793), HA (Cell Signaling Technology, #3724), H3K27ac (Cell Signaling Technology, #8173), p300 (Cell Signaling Technology, #54,062), YY1 (Cell Signaling Technology, #46395S), β-actin (Sigma, A2228), and the anti-rabbit and anti-mouse secondary antibodies (LI-COR, Ride® 800CW).