Fig 1: SsD inhibits patient primary cells. (A) CCK-8 assays in the cells derived from two patients treated with SsD for 48 h. (B) Colony-forming assays of patient cells treated with SsD. Graphs showing the colony number from 3 independent experiments. Data are the mean ± SD; *P < 0.05, **P < 0.01. (C) Effect of SsD on cell-cycle arrest was determined using FACS based on PI staining in patient cells after 48 h of treatment. (D) After 48 h of SsD treatment, m6A abundance in the RNA samples was determined in primary patient cells using a dot blot assay. (E) TSQ traces of FTO demethylation of m6A in ssRNA in the absence and presence (1 µM SsD for 48 h) of SsD in patient cells. (F) qPCR expression analysis of the indicated genes in patients' SsD-treated cells. Data represent three independent experiments. (G) Western blot analysis of the indicated genes in patients' SsD-treated cells. (H) Leukemia-bearing mice were developed by intravenous injection of patient cells into 4-week old NSG mice. (I) WBC count of leukemia-bearing mice (n = 3) is shown. (J) Images show representative external views of the spleen with the histological analysis of H&E-stained spleen sections (Scale bars, 1 cm, middle), Wright-Giemsa-stained bone marrow (BM) cells (left), and external views of the lung with H&E-stained sections (right) from leukemia-bearing mice. (K-M) The quantification of blast cells (K), spleen weight (L), and tumor growing nodules on lung (M) are shown. (N) The survival curve of leukemia-bearing mice calculated by Kaplan-Meier survival analysis (n = 5) is shown.
Fig 2: SsD induces m6A modification. (A) m6A dot blots of indicated cells treated with SsD are shown. MB (methylene blue) represents the loading control of RNA samples. (B) TSQ traces of FTO demethylation of m6A in ssRNA in the absence and presence of the SsD in NB4 and Kas-1 cells are shown, respectively. (C) Expression levels of indicated genes in cells treated with SsD measured using qPCR. Data represent three independent experiments. (D) Western blot analysis of the protein levels of indicated genes in SsD treated cells. (E-F) qPCR analysis of NB4 and Kas-1 cells treated with SsD for 24 h, followed by 5 µg/mL actinomycin-D treatment for the indicated time points. Gene expression was normalized to GAPDH. (G-H) Western blot analysis for protein stability in cells treated with SsD for 24 h, followed by 200 nM puromycin treatment for the indicated time points. Graphs are showing the quantification of Western blot analysis normalized to ß-actin; Data represent three independent experiments and present the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 3: FTO is a direct target of SsD. (A) The molecular structure of SsD is shown. (B) Molecular docking illustrations showing the potential binding pocket of SsD in FTO. (C) Structural complex of FTO bound with SsD. The surrounding amino acids and a-KG are shown. (D) Western blots showing the effects of 1 µM SsD on the thermal stabilization of FTO protein. CETSA was assayed in cell lysates. The results were derived from three biological replicates. (E) NMR measurement of SsD interaction with FTO. (F) Determination of cellular uptake of SsD by LC-MS/MS quantitation. AML cells were treated with 1 µM SsD for 48 h. (G) In vitro quantification of inhibition by SsD on FTO demethylation activity of m6A in RNA using HPLC. (H) In a cell-free system, dot blot analysis of m6A abundance in the presence of various SsD concentrations and FTO protein is shown.
Fig 4: Identification of genes and pathways related to the SsD response. (A) Representative scatter plot of 7174 genes in control vs SsD-treated NB4 cells, 3732 upregulated genes are marked in red while 3442 downregulated genes are marked in blue. (B) KEGG pathway analysis of both downregulated and upregulated genes (based on the RNA-seq results) in control vs SsD-treated NB4 cells. P-value was corrected by FDR, and the FDR < 0.01 was considered significantly enriched. (C) Similarly, Gene Set Enrichment Analysis (GSEA) of pathways for the downregulated and upregulated genes in control vs SsD-treated NB4 cells. The P-value was corrected by FDR, and the FDR < 0.01 was considered significantly enriched. (D) Venn diagram showing the core genes of SsD-suppressed signaling pathways and the four shared signaling pathways in MSigDB database. (E) The top four signaling pathways suppressed by SsD in NB4 cells. (F) Venn diagram showing up- or downregulated genes in signaling pathways after SsD treatment or knockdown-mediated FTO inhibition. (G) Heat map analysis for significantly down-regulated or up-regulated gene in SsD treated NB4 cells.
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