Fig 3. METTL3 knockout in U2OS cells also appears to be incomplete.

(A) METTL3 KO U2OS cells have persistent m⁶A. METTL3 KO U2OS cells have been reported to have 60% the levels of m⁶A found in control U2OS cells [24]. We reconfirmed this with mass spectrometry measurements of m⁶A, which showed that METTL3 KO U2OS cells have 75.2% remaining m⁶A compared to WT. Thus, m⁶A levels remain high in METTL3 KO U2OS cells. Error bars indicate standard error (n = 3). * = p-value < 0.5, ** = p-value < 0.01, *** = p-value < 0.005, n.s. = not significant. Underlying data can be found in S1 Data. (B) METTL3 KO U2OS cells express a novel anti-METTL3-antibody-reactive protein. As m⁶A levels were not completely ablated in the METTL3 KO U2OS cells, we assessed METTL3 protein expression in these cells to confirm if the knockout was effective. We found that WT METTL3 protein was lost in the METTL3 KO U2OS cells, but a larger protein that was reactive to the anti-METTL3 antibody was found in the METTL3 KO U2OS cells. This suggests the METTL3 KO U2OS cells express a novel METTL3 protein that is slightly larger than the WT METTL3. (C) Confirmation of a novel METTL3-like protein in METTL3 KO U2OS using a second METTL3 antibody. To confirm that the METTL3-immunoreactive band we saw in METTL3 KO U2OS cells in Fig 3B was METTL3, we used a second anti-METTL3 mAb to confirm the result. The same protein band is immunoreactive to the second anti-METTL3 antibody, thus suggesting that the METTL3 KO U2OS cells express a novel, larger METTL3 protein. (D) A METTL3-specific inhibitor leads to loss of m⁶A even in METTL3 KO U2OS cells. WT and METTL3 KO U2OS cells were treated with 30 μM STM2457, and m⁶A levels were measured by mass spectrometry after 48 h. m⁶A was reduced by 89.8% in the WT U2OS cells and 92.1% in the METTL3 KO U2OS cells after STM2457 treatment. It should be noted that 30 μM may not fully inhibit METTL3, so some of the residual m⁶A after STM2457 treatment may still derive from METTL3 isoforms. Thus, a METTL3 isoform is responsible for most of the remaining m⁶A in the METTL3 KO U2OS cells. Error bars indicate standard error (n = 3). * = p-value < 0.5, ** = p-value < 0.01, *** = p-value < 0.005, n.s. = not significant. Underlying data can be found in S1 Data. mAb, monoclonal antibody; m⁶A, N⁶-methyladenosine; pAb, polyclonal antibody; WB, western blot; WT, wild-type.