Ab195352

The harvested gastric mucosal tissues or GC tissues were fixed.
²² (link) The slides were incubated with the corresponding antibodies against METTL3 (ab195352, Abcam), HIF‐1α (sc‐13515, Santa Cruz), 4‐hydroxynonenal (4‐HNE, ab46545/ab48506, Abcam), Drp1 (8570S/ab156951, Cell Signalling Technology/Abcam), Ki67 (ab16667, Abcam), IGF2BP3 (14642‐1‐AP/ab177477, Proteintech/Abcam), LDHA (19987‐1‐AP, Proteintech) and NLRP3 (ab263899, Abcam). Immunofluorescence (IF) staining of the samples was performed using primary antibodies against Fis1 (A5821, ABclonal), Drp1 (8570S/ab156951, Cell Signalling Technology/Abcam), METTL3 (ab195352, Abcam), TOMM20 (ab186735/ab283317, Abcam), cytokeratin 18 (CK18, GTX105624, GeneTex), IGF2BP3 (14642‐1‐AP, Proteintech), NLRP3 (ab263899, Abcam) and interleukin (IL)‐1β (ab283818, Abcam). For double IF staining, a secondary targeted protein was examined after the initial protein detection stage. Nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole dihydrochloride (DAPI). Gastric sections were utilised for hypoxic status analysis with a Hypoxyprobe‐1 kit (Hypoxyprobe, Inc.). Transmission electron microscopy (TEM, Hitachi, H‐800) was used to examine gastric tissues or cell samples from a randomly selected pool of five fields. The length, width and area of the mitochondria were measured by ImageJ.

TMA was constructed from 90 pairs of formalin-fixed, paraffin-embedded renal cancer tissues and matched normal tissues (Tufei Biotech, Shanghai, China). Demographics and clinical characteristics of these samples are listed in Supplementary Table S2. After antigen retrieval, TMAs were blocked and stained with anti-METTL3 antibody (ab195352, abcam, UK). The degree of immunostaining was evaluated by two independent pathologists. IHC score was calculated as staining intensity (0 = negative, 1 = weak staining, 2 = moderate staining, 3 = strong staining, 4 = severe staining) multiplying staining area (0 = 0%, 1 = 1–25%, 2 = 26–50%, 3 = 51–75%, 4 = above 75%).
For IHC staining, tissues were fixed in 10% (v/v) formaldehyde in PBS, embedded in paraffin, and cut into 4 μm sections. Slides were incubated with specific primary antibodies against METTL3 (1:50, ab195352, Abcam, UK), PLOD2 (1:500, 21214-1-AP, Proteintech, USA), HIF-1α (1:100, ab51608, Abcam, UK) and Ki-67 (1:400, ab16667, Abcam, UK) respectively, at 4 °C overnight. Thereafter, secondary antibodies were added followed by DAB staining and haematoxylin counter-staining.

To generate the inducible PiggyBac donor vector with an amino-terminal FLAG tag fused to METTL3, we first synthesized gBlock (IDT) containing the BamHI-1XFLAG-METTL3-BamHI sequence. This fragment was then cloned into the BamHI entry site in the enhanced piggyBac transposable vector epB-Bsd-TT via cut-ligation to generate the vector, ePB-1xFLAG-METTL3 (Rosa et al. 2014 (link)).
Mettl3 KO mESCs were transfected with an equimolar amount of ePB-1xFLAG-METTL3 and piggyBac transposase (Rosa et al. 2014 (link)). Two days after transfection, cells were plated at clonal density and subjected to a transient puromycin selection (1 µg/mL) for 7 d. Colonies were picked 12 d after transfection, and western blot against endogenous METTL3 (Abcam, ab195352) was performed to identify clones which maintain comparable METTL3 expression between WT mESC cells and the ePB-METTL3 mESC line that was generated from parental Mettl3 KO mESCs. For experiments involving the ePB-METT3 ESC line, all inductions were performed using 100 ng/mL doxycycline (Sigma-Aldrich). A western blot of induced and uninduced cells, probed for METTL3 (Abcam, ab195352) and histone H3 (Abcam, ab1791), is shown in Supplemental Figure 3B. For the m6A-ELISA, mESC lines were cultured in 2i + LIF conditions on gelatin 0.1%-coated plates. Cells were harvested with Accutase (A1110501, Life Technologies).