αmettl3

Antibodies used in this study include αm⁶A (Millipore, ABE572 or MABE1006) for MeRIP/m⁶A-seq and MeRIP-qPCR, αHMBOX1 (Novus Biologicals, NBP1-31316) for ChIP-qPCR and immunoblotting, αγH2AX (JBW301, Millipore, 05-636) for immunofluorescence-FISH and Western blot, αTPP1 (Proteintech, 25849-1-AP) for immunoprecipitation (IP) and immunoblotting, and αTRF2 (Proteintech, 66893-1-Ig) for immunofluorescence-FISH. Other antibodies that were applied in Western blot analysis include αMETTL3 (Proteintech, 15073-1-AP), αMETTL14 (Sigma-Aldrich, HPA038002), αALKBH5 (Sigma-Aldrich, HPA007196), αHA (16B12) (BioLegend, #901501), αmyc (9E10) (Santa Cruz Biotechnology, sc-40), αHis (Proteintech, #66005-1), αCRISPR-Cas9 (Abcam, ab191468), αAR (H-280) (Santa Cruz Biotechnology, sc-13062), αYTHDF2 (Proteintech, 24744-1-AP), αYTHDC2 (Proteintech, 27779-1-AP), αp53 (DO-1) (Santa Cruz Biotechnology, sc-126), αp21 (Cell Signaling Technology, #2947), αMDM2 (Proteintech, 19058-1-AP), αTelomerase reverse transcriptase (TERT) (Abcam, ab32020), and αβ-actin (Sigma-Aldrich, A5441). Reagents that were used in IP, ChIP, and MeRIP are Protein A/G Plus Agarose (Santa Cruz Biotechnology, sc-2003) and Dynabeads Protein A (Thermo Fisher Scientific, #10006D) or Protein G (Thermo Fisher Scientific, #10007D) immunoprecipitation kit.

For performing western blot analysis, proteins were transferred onto a nitrocellulose membrane (GE Healthcare) using Towbin blotting buffer (192 mM glycine, 25 mM Tris/HCl pH 8.6, 20% methanol). Membranes were blocked in 1× TBST (150 mM NaCl, 10 mM Tris/HCl pH 8, 0.1% Tween 20) containing 5% skimmed milk for 1 h at 4 °C. After incubation with first and secondary antibody, the membrane was washed three times with 1× TBST. α-αTubulin (mouse, Sigma, clone DM1A) and α-METTL3 (rabbit, Proteintech Europe) were used as primary and α-mouse and α-rabbit (Licor) were used as secondary antibodies. The documentation was conducted using the Odyssey scanner system (LI-COR Biosciences).