Figure 1: Deletion of Alkbh4 causes embryonic lethality in mice and ALKBH4 associates with the contractile ring and midbody.

(a) Schematic diagram showing human (upper panel) and mouse (lower panel) ALKBH4 protein structure with conserved catalytic amino acids within the dioxygenase domain (upper panel) and deleted exons in the Alkbh4 knock-out allele (lower panel). (b) There were no homozygous Alkbh4^−/− offspring from heterozygous Alkbh4^+/− intercrosses. Mendelian distribution of WT and knock-out alleles indicated by the ratio 1:2:(1), WT: Het:(KO). +: WT allele; −: knock-out allele. (c) Endogenous ALKBH4 immunostaining in MRC5 cells with anti-ALKBH4 and F-actin staining with phalloidin. DNA was visualized by DAPI. Scale bars, 10 μm. Quantification of signal intensity (accumulation of protein) on the contractile ring (CR) and midbody (MID) relative to the whole cell (lower panel). (50 cells per condition per experiment). In box-and-whisker plot plotted by Prism5 software, median means 50% of cells is greater than this value, upper quartile or lower quartile means 25% cells is greater or less than this value and maximum or minimum means greatest or least value. (d) MRC5 cells stably expressing 3 × Flag-ALKBH4 were fixed and immunostained with anti-Flag and Phalloidin (F-actin). DNA was visualized by DAPI. Scale bars, 10 μm. Quantification of signal intensity (accumulation of protein) on the contractile ring (CR) and midbody (MID) relative to the whole cell (lower panel). (50 cells per condition per experiment). (e) MRC5 cells were fixed and immunostained with anti-ALKBH4 and anti-anillin (midbody marker). DNA was visualized by DAPI. Scale bars, 10 μm. Quantification of signal intensity (accumulation of protein) on the contractile ring (CR) and midbody (MID) relative to the whole cell (lower panel). (50 cells per condition per experiment). (f) MEF cells were fixed and immunostained with antibodies against ALKBH4 and α-tubulin (α-tub). DNA was visualized by DAPI. (g) MRC5 cells were sequentially synchronized with thymidine and nocodazole; and whole-cell extracts (WCE) and contractile ring/midbody (CM) fractions were analysed by immunoblotting with the indicated antibodies. Full-length blots are presented in Supplementary Fig. S7. Scale bars in all panels, 10 μm.