Si dnmt1

Conditionally immortalized mouse podocytes (MPC-5) and human HEK293 cells (h243) were obtained from iCell Bioscience Inc. (Shanghai, China). Podocytes and HEK293 cells were grown in Dulbecco’s modification of Eagle’s medium (DMEM) (GIBCO, New York, USA) containing 10% fetal bovine serum (GIBCO), 1% penicillin-streptomycin, and incubated at 37°C with 5% CO
₂. After cell cultures reached a confluence of 70%--80%, podocytes were treated with 80 μM Hcy (Sigma-Aldrich, Darmstadt, Germany) for 48 h. For transfection experiments, miR-30a-5p inhibitor, miR-30a-5p mimic, miRNA negative control (miR-neg), and small interfering RNAs (siRNAs) specifically targeting DNA methyltransferase enzyme 1 (si-DNMT1) were obtained from Gene Pharma (Shanghai, China). Recombinant adenoviruses expressing DNMT1 (Ad-DNMT1) were purchased from HANBIO (Shanghai, China). Then, the cells were seeded in 6-well plates and transfected according to the supplier′s instructions. The siRNA sequences of DNMT1 are as follows: si-NC: 5′-UUCUCCGAACGUGUCACGUTT-3′; si-DNMT1-1: 5′-GCAAAGAGUAUGAGCCAAUTT-3′; si-DNMT1-2: 5′-GCUGGUCUAUCAGAUCUUUTT-3′, and si-DNMT1-3: 5′-CCGAGGCCUUUACUUUCAATT-3′.

The cells were transfected using Lipofectamine 2000 (11 668 030, Invitrogen Inc., Carlsbad, CA, USA) as per the manufacturer’s instructions. The siRNA targeting DNMT1 mRNA (si-DNMT1), DNMT3A mRNA (si-DNMT3A), DNMT3B mRNA (si-DNMT3B), RARB mRNA (si-RARB) and negative control (si-NC) were acquired from Shanghai GenePharma Co., Ltd. (Shanghai, China). The sequences of the siRNAs are shown in Table 1. The full-length RARB cDNA was subcloned into pcDNA3.1 vector (Invitrogen) for the construction of RARB overexpression (oe-RARB), with the empty vector as a negative control (oe-NC). After seeding and plating in 6-well plates for 1 d, the cells were transfected with the plasmids. After 48 h of transfection, the cells were stored for later use. Transfection efficiency was determined by RT-qPCR.
To suppress the expression of DNMT1, the cells were treated with 10 μM 5-aza-2′-deoxycitidine (5-AzaC) (189 825, Sigma-Aldrich Chemical Company, St Louis, MO, USA) 3 days before transfection. An equal volume of dimethylsulfoxide (DMSO) was applied as a control.

The human normal bone marrow stromal HS5 cell and AML cell lines (MOLM-16, NB-4, HEL 92.1.7, HEL, EOL-1) were procured from reputable sources such as the American Type Culture Collection (ATCC) or the Shanghai Cell Bank (Type Culture Collection, Chinese Academy of Sciences). These cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin, 50 mg/ml streptomycin, and 2 mmol/L glutamine. The cells were maintained in a humidified CO₂ incubator at 37 °C. To ensure the integrity of the cell lines, all cells were passaged for a duration of less than 3 months, and renewal was done using frozen, early-passage stocks obtained from the aforementioned sources. The small interfering RNAs (siRNAs), namely si-Con, si-DNMT1, and si-ATR, were synthesized by Gene-Pharma (Shanghai, China). Lipofectamine 3000 (Invitrogen) was employed for cell transfection in accordance with the guidelines provided by the manufacturer. DNMT1 inhibitor (GSK3685032 or GSK-368), ATM inhibitor (AZD-1390) and ATR inhibitor (AZD-6738) were purchased from MedChemExpress (Shanghai, China).