Fig. 1: METTL3 is highly O-GlcNAcylated in HCC. | Cell Death & Disease

Fig. 1: METTL3 is highly O-GlcNAcylated in HCC.

From: O-GlcNAcylation of METTL3 drives hepatocellular carcinoma progression by upregulating MCM10 expression in an m6A-IGF2BP3-dependent manner

Fig. 1

A Huh-7 hepatoma cells were transfected with Flag-METTL3, Flag-METTL14, or Flag-WTAP for 48 h. Cell lysates were collected for sWGA pull-down assays to identify O-GlcNAcylation. METTL3 O-GlcNAcylation levels in HCC tumors (T) and paired adjacent non-tumor (NT) tissues were detected by sWGA pull-down assays (B), quantified using ImageJ and analyzed with a two-tailed paired Student’s t-test (C), P < 0.001. D, E Analysis of METTL3 O-GlcNAcylation using chemoenzymatic labeling in Huh-7 and PLC/PRF/5 hepatoma cells treated with 25 μM TMG for 12 h. F, G Huh-7 and PLC/PRF/5 cells were treated with 25 μM TMG or 20 μM OSMI-1 for 12 h. Then, cell lysates were pulled down with sWGA-conjugated agarose and immunoblotted with anti-METTL3. H, I Huh-7 and PLC/PRF/5 hepatoma cells were transfected with Flag-METTL3 or a control vector for 48 h. After treatment with 25 μM TMG for 12 h, cell lysates were immunoprecipitated using anti-Flag M2 agarose beads. J For the in vitro O-GlcNAcylation assay, recombinant His-METTL3 proteins and enzymatic GST-OGT domain (aa 313–1031) were incubated in reaction buffer for 4 h. Immunoblot analyses and Coomassie blue staining were performed.

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