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Fig. 5 | Journal of Experimental & Clinical Cancer Research

Fig. 5

From: The essential roles of m6A RNA modification to stimulate ENO1-dependent glycolysis and tumorigenesis in lung adenocarcinoma

Fig. 5

M6A methylation of ENO1 mRNA was critical for its translation and glycolysis.(A) Representative IB images of ENO1 in H1650 and H1975 cells treated with DMSO or DAA (100 μM, 24h). (B) Polysome profiling in H1975 cells with or without combined METTL3 knockout and ALKBH5 overexpression. (C) Ribosome-associated ENO1 mRNA in H1975 cells with or without combined METTL3 knockout and ALKBH5 overexpression. (D) Polysome profiling in H1299 cells with or without combined ALKBH5 knockout and METTL3 overexpression. (E) Ribosome-associated ENO1 mRNA in H1299 cells with or without combined ALKBH5 knockout and METTL3 overexpression. (F) Prediction and verification of potential m6A sites within ENO1 mRNA, as predicted by SRAMP online software and verified by RIP experiments using anti-m6A antibodies. (G) ENO1 protein expression and translation efficiency in ENO1-/- H1299 cells that reconstituted with WT or Mut ENO1 (359A to 359G), with or without combined ALKBH5 knockout and METTL3 overexpression. (H) Schematic presentation of the construction of the pmir-GLO-ENO1 reporter containing ENO1 partialORF region with or without 359A mutation. (I) Translation efficiency of ENO1-LUC fusion mRNA, as calculated by the ratios between luciferase activities and mRNA levels in H1299 cells with or without combined ALKBH5 knockout and METTL3 overexpression. (J-L) ENO1 activity (J), PEP (J), glucose uptake (J), lactate production (J), ATP (J), ECAR (K) and OCR (L) in ENO1-/- H1299 cells reconstituted with WT or Mut ENO1 (359A to 359G), with or without combined ALKBH5 knockout and METTL3 overexpression. (M-O) Representative images (M), number (N) and size (O) of 3D-spheroids that generated by ENO1-/- H1299 cells reconstituted with WT or Mut ENO1 (359A to 359G), with or without combined ALKBH5 knockout and METTL3 overexpression. Scale bar, 100 μm. Statistical analysis was performed using t test (C, E), one-way ANOVA (G, I, J, N, O) and two-way ANOVA (K, L). Data are presented as means ± SEMs from three independent experiments. **p < 0.01, *p<0.05 indicates statistical significance and N.S. indicates no significance

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