Fig. 4: SETBP1 was a direct target of METTL14-mediated m⁶A modification in MDS.

A Obvious decrease of SETBP1 mRNA in MDS-L cells with METTL14 knockdown indicated by q-PCR analysis. ***P < 0.001. B Western blot showing METTL14 knockdown led to significant decrease of SETBP1 protein in MDS-L cells. C Noticeable increase of SETBP1 mRNA in MDS-L cells with overexpression of METTL14 WT, but not the METTL14 R298P, indicated by q-PCR analysis. ***P < 0.001. D Western blot showing forced expression of METTL14 WT, but not the METTL14 R298P, increased SETBP1 protein. E MeRIP-qPCR showing a remarkable reduction of m⁶A modification in specific regions of SETBP1 mRNA transcripts upon METTL14 knockdown. ***P < 0.001. F MeRIP-qPCR showing an increase of m⁶A modification in MDS-L cells with overexpression of METTL14 WT, but not the METTL14 R298P. **P = 0.001. G RIP-qPCR showing METTL14 protein bound to SETBP1 mRNA in MDS-L cells. ***P < 0.001. H RNA stability assays showing that METTL14 knockdown decreased the half-life of SETBP1 mRNA. ***P < 0.001. I Dual-luciferase assays of SETBP1 WT 3’-UTR-reporter showing the decreased activity of luciferase upon METTL14 knockdown, while METTL14 knockdown had no effect on SETBP1 3’-UTR-reporter with mutated m⁶A sites. **P = 0.008 (left), 0.001 (right). J MeRIP-qPCR showing a significant reduction of m⁶A modification in specific regions of SETBP1 mRNA transcripts upon METTL3 knockdown. **P = 0.002; ***P < 0.001. K Significant decrease of SETBP1 mRNA in MDS-L cells with METTL3 knockdown indicated by q-PCR analysis. **P = 0.002 (left), 0.001 (right). L Western blot showing METTL3 knockdown led to significant decrease of SETBP1 protein in MDS-L cells. M Co-IP experiments showing the overexpression of METTL14 led to an increase in the binding with METTL3. N Western blot showing the overexpression of METTL14 did not alter the overexpression of METTL3. O MeRIP-qPCR showing a remarkable reduction of m⁶A modification in specific regions of SETBP1 mRNA transcripts treatment with STM2457 (10 μM) for 48 hours. **P = 0.003. P Obvious reduce of SETBP1 mRNA in MDS-L cells treated with STM2457 (10 μM) for 48 hours indicated by q-PCR analysis. ***P < 0.001. Q Western blot showing treatment with STM2457 (10 μM) for 48 hours led to significant decrease of SETBP1 protein in MDS-L cells. R CellTiter-Lumi^TM assay showing that treatment with STM2457 for 48 hours resulting in concentration-dependent inhibition of cell viability of MDS-L cells, with an IC₅₀ of 6.87 μM. S, T Chemiluminescence imaging (S) and its luminescence counts (T) showing a noticeable suppression of MDS-L-luc cells engraftment in mice after treatment of STM2457 (50 mg/kg). *P = 0.034. U Flow cytometry showing that treatment of STM2457 (50 mg/kg) remarkably decreased the percentages of human CD45⁺ cells in bone marrow of mice. ***P < 0.001. V Kaplan–Meier survival analysis showing that treatment of STM2457 (50 mg/kg) in vivo prolonged the OS of mice. The experimental data were analyzed with Student’s t-test. Error bars denoted mean ± SD of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; N.S No significance.