Alkbh5

Mouse pancreas was collected and fixed in 4% formaldehyde at 4°C overnight, followed by paraffin embedding. Five-micron thick slides were cut and subjected to immunostaining. Slides were heated in 10mM sodium citrate, followed by blocking with donkey serum and incubated with various primary antibodies: Ki67 (#550609, BD, USA), Anti-METTL3 (#195352, Abcam, USA), Anti-METTL14 (#HPA038002, Sigma, USA), ALKBH5 (#HPA007196, Sigma, USA), PDX1 (#5679, Cell Signaling, USA), Insulin (#ab7842, abcam, USA), Glucagon (#G2654, Sigma, USA), Somatostatin (#ab64053, abcam, USA). Specific signal was detected by using fluorescence-conjugated secondary antibodies (Jackson Immunoresearch, Alexa 488, Alexa 594, and AMCA) (please see Reporting Summary for details on antibodies used). Images were captured using Zeiss Axio Imager A2 upright fluorescence microscope. The β-cell mass was calculated by generating the ratio of the cross-sectional area of total number of pixels of Insulin positive cells to the cross-sectional area of total number of pixels of the pancreatic tissue, multiplied by the pancreas weight of the mouse. We evaluated β-cell proliferation by co-immunostaining one section of each pancreas sample with Ki67, Insulin and DAPI and counting 1000–2000 cells in a blinded manner by a single observer^{33 (link),34 (link),37 (link)}.

Cells were washed twice with ice-cold phosphate-buffered saline (PBS) and ruptured with RIPA buffer (Pierce, Rockford, IL) containing 5 mM EDTA, PMSF, cocktail proteinase inhibitors, and phosphatase inhibitor cocktail. Cell extracts were centrifuged at 12000 × g for 15 min and the supernatants were then collected. Cell lysates were resolved by SDS-PAGE and transferred onto PVDF membranes which were blocked with 5% non-fat milk (Bio-Rad) in Tris-buffered saline containing 0.1% Tween 20 for 1 hour and incubated sequentially with primary and secondary antibodies and detected by immunoblotting with the Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) or Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare). Antibodies used for Western blotting were as follows: ALKBH5 (ab195377, abcam), ALKBH5 (HPA007196, Sigma-Aldrich), TACC3 (sc-376883, Santa Cruz Biotechnology), Flag (F3165, Sigma-Aldrich), c-Myc (9402S, Cell Signaling Technology), P21 (2947S, Cell Signaling Technology) GAPDH (sc-47724, Santa Cruz Biotechnology), β-Actin (3700, Cell Signaling Technology), Vinculin (sc-25336, Santa Cruz Biotechnology). GAPDH or β-Actin or Vinculin was used as a loading control.