Cell lines and cell culture

KG1a, a myeloid leukemia cell line, and SKM1, a cell line derived from MDS that progresses to AML, were grown and propagated as described previously [19]. Briefly, KG1a and SKM1 cells were cultured in RPMI 1640 medium (HyClone; Provo, UT, USA) containing 10% fetal bovine serum (FBS; Biological Industries, Beit Haemek, Israel) at 37 °C in a 5% CO2 atmosphere.

Transfectant construction

The full-length FTO, OGT and OGA genes were cloned from cDNA of KG1a cells. Site-directed mutagenesis was performed using fusion PCR. The wild type and the site-specific mutant FTO were individually inserted into the pLVX-AcGFP-N1 plasmids (Takara; Shiga, Japan). Lentiviral vectors were constructed and packaged into HEK-293T cells using the packaging system, pMD2.G and psPAX2, along with polyethylenimine MAX (PEI, Polysciences Inc.; Warrington, PA, USA). Stable transfectants were established by infecting cells with the lentivirus and selecting them using puromycin (Sigma-Aldrich). All siRNA fragments used in this study were purchased from Beijing Tsingke Biotechnology. For siRNA transfection, we followed the instructions for the HighGene Plus Transfection Reagent (ABclonal Technology, Wuhan, China). Briefly, siRNA at a final concentration of 100 nM was mixed with the transfection reagent and added to the cells. After 48 h, RT-qPCR or western blotting was performed for analysis.

Patient samples

Mononuclear cells were isolated from bone marrow aspirates of patients with MDS/AML and separated by gradient centrifugation through Ficoll-Hypaque (TBD, Tianjin, China) [20]. CD34+ cells were sorted using a CD34 microsphere kit (Miltenyi Biotechnology, Bergisch Gladbach, Germany) [21]. CD34+ cells were treated with 20 µM DMSO and 20 µg/mL OSMI-1 (MCE, NJ, USA) for 48 h, respectively. Written informed consent, in accordance with the Declaration of Helsinki, was obtained from all patients. All protocols were reviewed and approved by the research ethics committee of Northwest University.

Proteomic analysis

Protein (500 µg) was denatured with 8 M urea, reduced with 5 mmol/L DTT (Sigma-Aldrich) for 1 h at room temperature (RT), alkylated with 20 mmol/L IAM (Sigma-Aldrich) for 30 min at RT in the dark, diluted with distilled water to reduce the urea concentration below 2 mol/L, digested with lysylendopeptidase (Wako Puro Chemical; Osaka, Japan) and trypsin (Promega; Madison, WI, USA). The peptide mixture was lyophilized, resuspended in 50 mmol/L NH4HCO3, and purified using Oasis HLB cartridges. Two-dimensional LC-MS were carried out using an LTQ Orbitrap MS (Thermo Fisher, San Jose, CA, USA). Data was analyzed using Proteome Discover (Thermo Fisher) and MaxQuant software program as described previously [22].

In vivo mouse experiment

Animal experiments were conducted in strict accordance with the guidelines of the Animal Care and Use Committee of Northwest University. Tissue-specific OGT-KO mice were generated using the Cre-LoxP system by crossing OGT-flox (OGTfl/fl) mice (Jackson Laboratory, Bar Harbor, ME, USA) with Mx1-Cre mice (Jackson Laboratory) to produce HSC OGT-KO mice. Cre activation was induced by intraperitoneal injection of 250 µg polyinosinic acid (poly I: C, Sigma-Aldrich), and the mice were humanely euthanized after six weeks. Following euthanasia, mononuclear cells were isolated from the femurs of OGTfl/fl and Mx1-Cre; OGTfl/fl mice and further purified using cKit+ magnetic beads (Miltenyi Biotec). Subsequently, the cells were infected with MLL-AF9 lentivirus (kindly provided by Professor Guangyao Kong, Xi’an Jiaotong University) to establish a primary AML cell model.

In vitro O-GlcNAcylation

In vitro O-GlcNAcylation was performed as described [23]. FTO fused with a 6 × His tag, and OGT fused with a GST tag, were individually overexpressed in E. coli BL21 (DE3) and purified. His-tagged FTO (10 µg), GST-tagged OGT (20 µg) and 2.5 mM UDP-GlcNAc (donated by Prof. Junqiang Fang, Shandong University, China) were added to a 100 µL reaction buffer (125 mM NaCl, 1 mM EDTA and 20 mM Tris HCl, pH 7.4), and reacted at 37℃ for 6 h. O-GlcNAc-modified FTO underwent SDS-PAGE separation and a series of treatments, including dehydration, reduction, alkylation, and trypsin digestion. The resulting peptides were separated using a PepMap C18 nano column on the ThermoFisher U3000 RSLCnano system (#QE HF-X, ThermoFisher, MA, USA). The O-GlcNAcylation sites were analyzed using pFind software [24].

Western blotting

The cells were collected, rinsed three times with cold PBS and lysed in RIPA buffer containing 50mM Tris (pH 7.2), 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 150 mM NaCl, 10 mM MgCl2 and 2.5% glycerol. Protein lysates were incubated on ice for 30 min and centrifuged at 14,000 g for 15 min at 4 °C. The supernatant was collected and assayed using BCA kit (Beyotime Biotechnology, Shanghai, China). Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Merck KGaA, Darmstadt, Germany). The membranes were blocked with 3% (w/v) bovine serum albumin (BSA; Sigma-Aldrich) in TBST (20 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.05% Tween 20, pH 8.0) for 1 h at 37 °C. Subsequently, the membranes were incubated with primary antibodies overnight at 4 °C, followed by incubation with the appropriate HRP-conjugated secondary antibodies (Beyotime). The bands were developed using an ECL solution (Vazyme Biotech, Nanjing, China) and photographed using a gel documentation system (Tanon Science & Technology). Bcl2 (15071, CST, Danvers, MA, USA). Anti-GAPDH (G-9545) was obtained from Sigma-Aldrich (St. Louis, MO, USA), Anti-O-GlcNAcylation (PTM BIO, Hangzhou, China) and anti-m6A (ab286164) were from Abcam (Cambridge, MA, USA). Anti-SOX4, Anti-OGT, Anti-OGA and HA-tag were from Abclonal.

Immunoprecipitation (IP)

KG1a cells were treated with 20 µM Thiamet G (TMG, MCE) and 20 µg/mL OSMI-1 respectively for 48 h. Cells were lysed with lysis buffer (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 150 mM NaCl, and 1% Triton X-100) containing Protease Inhibitor Cocktail (4693159001, Roche, Switzerland) and Phosphatase Inhibitor (NCM Biotech, Co, Ltd). After centrifugation, the supernatant was incubated with anti-HA antibody (ABclonal) at 4 °C overnight, centrifuged and collected for further use.

Protein co-immunoprecipitation (Co-IP)

Cell lysates underwent incubation with anti-FTO antibody (Proteintech, Wuhan, China) overnight at 4 °C, followed by treatment with protein A/G agarose (Santa Cruz Biotechnology, TX, USA) for a duration of 12 h at 4 °C. Subsequently, the agarose was subjected to PBS washing, harvested through centrifugation at 2, 000 g for 5 min, and the samples were subjected to boiling in 1 × SDS Sampling Buffer for a period of 10 min prior to Western blotting.

m6A dot blot

Total RNA was isolated using the Trizol method (ABclonal). The concentration and purity of the isolated RNA were measured with a DS-11 spectrophotometer (DeNovix Inc.). The total RNA was then denatured at 95 °C for 3 min and spotted dropwise onto a positively charged nylon membrane (Beyotime), which had been pre-blocked with 5% skimmed milk. The membrane was incubated overnight at 4 °C with an anti-m6A antibody (Abcam, 1:2,000). On the following day, an HRP-conjugated anti-rabbit IgG secondary antibody (Beyotime) was applied to the membrane and incubated with gentle shaking for 1 h at room temperature. Enhanced chemiluminescence was used for signal development. Methylene blue staining was performed to confirm equal loading of total RNA on the membrane. The dot blot analysis was performed using a total amount of 400 ng for quantitative analysis.

Cell apoptosis

1 × 106 KG1a cells were harvested, washed twice with cold PBS and suspended in 1 mL binding Buffer (BioLegend, San Diego, CA, USA). Then 5 µL each of Annexin V and 7-AAD (BioLegend) will be added simultaneously to 100 µL of cell suspension. The cell suspension was vortexed and allowed to incubate for 15 min in the dark at room temperature. 400 µL of 1 × binding buffer was then added to each tube and analyzed by flow cytometry.

Cell proliferation

Cells were treated with 10 mmol/L EdU (GeneCopoeia; CA, USA) for 4 h. Cells were collected and fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton-X100, and individual samples were incubated with reagents containing 2 µL of Cu2SO4, 0.5 µL of Alexa Fluor 647 azide, 1 µL of 10 × Reaction buffer additive and 96.5 µL of PBS, mixed well and added to the treated cells, and stained for 30 min at room temperature away from light. Flow cytometry was used to detect EdU fluorescence signals.

Cell cycle

1 × 106 KG1a cells were washed with cold PBS, fixed with 75% cold ethanol and store overnight at 4 °C. After rinsing with cold PBS, resuspend fixed cells with 0.5 mL PBS, add 100 µg/mL RNase A (YEASEN, Shanghai, China) and 50 µg/mL PI (BioLegend), and incubate at 37 °C for 1 h. Flow cytometry is used for fluorescence signal detection.

Protein stability assay

To test the stability of O-GlcNAc modification on FTO protein, KG1a cells were treated with 20 µM TMG and 20 µg/mL OSMI-1 for 12 h and then added 50 µM cycloheximide (CHX, MCE) to continue the treatment and cells were collected at 0, 3, 6 and 9 h.

mRNA stability assay

Cells were treated with 5 µg/mL actinomycin D (S8964, Selleck, TX, USA) for 0, 4, 8, and 12 h, and total RNA was extracted with Trizol. mRNA levels were analyzed by real-time quantitative PCR (RT-qPCR). mRNA half-life (t1/2) was calculated using the following formula: t1/2 = In 2/kdecay [25].

RNA immunoprecipitation and sequencing (RIP and RIP-seq)

Cells were lysed with RIP buffer and subjected to freeze-thaw cycles. Following centrifugation, the supernatant was mixed with anti-FTO antibody-conjugated protein A/G magnetic beads and allowed to incubate for 4–6 h. Subsequently, the immunoprecipitated material was eluted, treated with proteinase K, and underwent RNA extraction using TRIzol. The extent of interaction between FTO and target RNA was assessed through RT-qPCR and normalized against the input. In the case of RIP-seq, rRNAs were removed utilizing the NEBNext rRNA Depletion Kit (New England BioLabs). Subsequently, cDNA libraries were generated and sequenced on the Illumina Novaseq™ 6000 platform from LC-BIO Technology Co.

Methylated RNA immunoprecipitation sequencing (m6A-seq)

m6A-Seq was performed by LC-Bio Technology Co., Ltd. (Hangzhou, China), according to manufacturer’s instructions. KG1a cells were treated with 20 µM DMSO and 20 µg/mL OSMI-1 for 48 h, respectively, and total RNA was purified and subjected to m6A RNA immunoprecipitation. Eluted m6A-containing fragments (IP) and untreated input control fragments were converted to the final cDNA library following strand-specific library preparation using the dUTP method. The average insert size for the paired-end libraries was approximately 100 ± 50 bp. Paired-end sequencing (PE150) was performed on an Illumina Novaseq™6000 platform.

MeRIP RT-qPCR analysis

Poly(A) mRNA was purified using the Dynabeads mRNA Purification Kit (Invitrogen). 10% of the RNA was retained as an input control. Protein A/G magnetic beads (Beyotime) were pre-washed and incubated with 5 µg anti-m6A antibody (Abcam) or rabbit IgG for 2 h at 4 °C with rotating. After washing of the conjugate beads, the antibody-conjugated beads were conjugated to the purified poly(A) mRNA and incubated overnight at 4 °C. The m6A RNA was then eluted with elution buffer and recovered by ethanol precipitation. Further enrichment was performed by qPCR and m6A enrichment calculated for each sample by normalization [26].

Data Analysis

Experimental data were statistically analyzed using GraphPad Prism 9.0 software, with experimental results repeated three times. Statistical comparisons between two groups were made using the unpaired t-test. Significant differences in this thesis are labelled as *P < 0.05; **P < 0.01; ***P < 0.001, respectively.