Fig. 2: METTL14 promotes MDS cell proliferation in vitro and in vivo.

A Western blot showing the knockdown effect of METTL14 in METTL14-knockdown MDS-L cells which generated through lentivirus. B Dot blot assay showing METTL14 knockdown led to obviously decreased m⁶A modification of MDS-L cells. C CellTiter-Lumi^TM assay showing that knockdown of METTL14 significantly inhibited the cell proliferation of MDS-L cells. ***P < 0.001. D The colony formation assay showing that knockdown of METTL14 markedly inhibited the colony formation of MDS-L cells. ***P < 0.001. E Flow cytometry showing that knockdown of METTL14 obviously increased the apoptosis of MDS-L cells. ***P < 0.001. F Flow cytometry showing that knockdown of METTL14 blocked the cell cycle in G0/G1 phase of MDS-L cells. ***P < 0.001; **P = 0.003 (left), 0.005 (right); *P = 0.014. G CellTiter-Lumi^TM assay showing that knockdown of METTL14 markedly inhibited the cell proliferation of CD34⁺ MDS blasts from a patient with EB-2. **P = 0.003 (left), 0.003 (right). H The colony formation assay showing that knockdown of METTL14 significantly inhibited the colony formation of CD34⁺ MDS blasts from a patient with EB-2. ***P < 0.001. I Western blot showing the overexpression effect of METTL14 in METTL14 WT and METTL14 R298P-overexpressing MDS-L cells. J Dot blot assay showing that overexpression of METTL14 WT, but not METTL14 R298P, increased m⁶A levels of MDS-L cells. K CellTiter-Lumi^TM assay showing that overexpression of METTL14 WT, but not METTL14 R298P, significantly enhanced the cell proliferation of MDS-L cells. ***P < 0.001. L The colony formation assay showing that overexpression of METTL14 WT, but not METTL14 R298P, significantly enhanced the colony formation of MDS-L cells. ***P < 0.001. M Western blot showing the knockdown effect of METTL14 in MDS-L cells expressing Dox-inducible shMETTL14 (shMETTL14_Tet-on) after 2 days of Dox treatment. N CellTiter-Lumi^TM assay showing that knockdown of METTL14 by a Dox-inducible shMETTL14 significantly inhibited the cell proliferation of MDS-L cells in vitro. ***P < 0.001. O In vivo experimental scheme for (O–T): briefly, NCG-M mice xenografted with the MDS-L-luc cells transduced with Dox-inducible shMETTL14 subjected to the Dox treatment or the vehicle. (P, Q) Chemiluminescence imaging (P) and its luminescence counts (Q) showing a noticeable suppression of MDS-L-luc cells engraftment in mice following Dox-induced knockdown of METTL14. ***P < 0.001; **P = 0.002. R, S Flow cytometry showing that Dox-induced knockdown of METTL14  in vivo remarkably decreased the percentages of human CD45⁺ cells in bone marrow (R) and peripheral blood (S) of mice. ***P < 0.001. T Kaplan–Meier survival analysis showing that Dox-induced knockdown of METTL14 in vivo prolonged the OS of mice. The experimental data were analyzed with Student’s t-test. Kaplan–Meier survival analysis was performed using log-rank test. Error bars denoted mean ± SD of three independent experiments. N.S No significance.