Cell lines and compounds
The HEK293T (CRL-3216), 4T1 (CRL-2539), MDA-MB-231 (HTB-26), MDA-MB-468 (HTB-132), MCF10A (CRL-10317), and HC11 (CRL-3062) cell lines were obtained from the American Type Culture Collection (ATCC). HEK293T, MDA-MB-231, MDA-MB-468, and MCF10A cells were maintained in DMEM (11965118, Gibco, USA), while 4T1 and HC11 cells were cultured in RPMI 1640 medium (11875119, Gibco). The 4T1-luc and MDA-MB-231-luc cell lines were constructed by infecting wild-type cells with luciferase-expressing lentiviruses (LV-NC009, Guangzhou IGE Biotechnology, Guangzhou, China). All the culture media were supplemented with 10% fetal bovine serum (FBS) (A5670701, Gibco, California, USA) and 1% penicillin–streptomycin (15140163, Gibco). Cells were maintained in a humidified incubator at 37 °C with 5% CO2. All the cell lines have been confirmed to be mycoplasma-free via PCR-based detection (Forward primer: 5’-GGGAGCAAACAGGATTAGTATCCCT-3’; Reverse primer: 5’-TGCACCATCTGTCACTCTGTTACCCTC-3’).
Decitabine (DAC) (T1508), erastin (T1765), RSL3 (T3646), Ferrostatin-1 (Fer-1) (T6500), LCL161 (T2080), and Cisplatin (T1564) were purchased from TargetMol Chemicals. BIX-01294 (BIX) (S8006) and mitomycin C (S8146) were purchased from Selleck Chemicals. All compounds were dissolved in DMSO, except that Cisplatin was dissolved in saline. The stock solutions of these compounds were prepared as follows: DAC, 100 mM; BIX, 100 mM; erastin, 10 mM; Fer-1, 50 mM; RSL3, 10 mM; LCL161, 50 mM; mitomycin C, 1 mg/mL; Cisplatin, 3 mM (in saline).
Microbial strains
Escherichia coli (E. coli) strains DH5α (KTSM101L, AlpaLifeBio, Shenzhen, China) and BL21 (DE3) (KTSM104L) were cultured at 37 °C in Luria-Bertani (LB) medium containing specified antibiotics. DH5α was utilized for propagating constructed protein-coding clones, while BL21 (DE3) was used for the expression and purification of prokaryotically expressed proteins, including GFP-tagged DNMT1-DS1 and DNMT1-DS1 mutants.
Cell viability assay
To evaluate the dose-dependent response of targeted compounds, about 10,000 4T1 cells, 5000 MDA-MB-231 cells, and 10,000 MDA-MB-468 cells were seeded into each well of the 96-well plates, respectively. Triplicate wells were prepared for all three independent experiments. Cells were incubated overnight at 37 °C with 5% CO2. On the next day, the culture medium was replaced with freshly prepared 100 μL of DMEM or 1640 culture medium containing different concentrations of DAC or BIX. The control wells were replaced with fresh medium containing DMSO. 4T1 and MDA-MB-468 cells were incubated for another 3 days, while the MDA-MB-231 cells were incubated for 7 days. Four hours (h) before detection, about 10 μL of Cell Counting Kit-8 (CCK-8) reagent (C0005, TargetMol) was added to each well, followed by incubaton for 4 h. The optical density (OD) value was measured at a wavelength of 450 nm using a multifunctional microplate reader (Tecan Spark, Männedorf, Switzerland). OD450 values were then curve-fitted using GraphPad Prism 9 software to calculate the half-maximal inhibitory concentration (IC50) of each compound in each cell line.
Evaluation of combination effects
To calculate the combination index (CI) value, the inhibitory effects on cell growth were assessed utilizing combinations of different concentrations of the DNMT1 inhibitor DAC and the G9a inhibitor BIX. For each treatment condition, the cell viability data were normalized to represent the fraction affected (Fa) value as a percentage relative to the untreated control. The CI values corresponding to different conditions were calculated using CompuSyn software. A CI value < 1 was defined as a synergistic effect. A CI value = 1 was defined as an additive effect, while a CI value > 1 was defined as an antagonistic effect.
Colony formation assay
4T1, MDA-MB-231, and MDA-MB-468 cells undergoing exponential growth were digested with 0.25% trypsin (25200114, Gibco) to obtain the single-cell suspension. Approximately 1000 cells were seeded in 6-well plates in triplicate. Three independent experiments were performed. On the next day, 4T1 cells were treated with 100 nM DAC and/or 1 μM BIX for 7 days. MDA-MB-231 cells were treated with 50 nM DAC and/or 100 nM BIX for 14 days. MDA-MB-468 cells were treated with 10 nM DAC and/or 10 nM BIX for 14 days. The control group was treated with equal volume of DMSO. Each treatment was conducted in triplicate. During the period of cultivation, the medium was replaced with freshly prepared compound-containing medium every 3 days. After the cultivation, cells were fixed with 4% paraformaldehyde for 20 min (min) and stained with 0.2% crystal violet for an additional 20 min. After washing with PBS, the plates were dried at room temperature. Images of each well were captured utilizing an inverted optical microscope (Sunny Optical Technology, Hangzhou, China). Cellular colonies within each well were subsequently quantified using ImageJ software.
Wound healing scratch assay
4T1, MDA-MB-231, and MDA-MB-468 cells in the exponential growth phase were seeded in 6-well plates at a density of 1 × 10⁶ cells per well, in triplicate. On the next day, a 10 μL pipette tip was used to generate a scratch in the cell monolayer, followed by washing the cells three times with PBS to remove scratched cells. To exclude effects of cell proliferation-mediated wound healing, cells were treated with 1 μg/mL mitomycin C for 1 h. Cells within each well were then treated with 1 μM DAC and/or 1 μM BIX, while the control group was treated with an equal volume of DMSO. Images of the scratched area were captured at 0 h and 48 h post-scratch using an inverted optical microscope (Sunny Optical Technology). The experiment was repeated three times. The formula for calculating the wound healing rate (%) was: [(0 h scratch width – 48 h scratch width) / 0 h scratch width] × 100%.
Transwell migration assay
TNBC cells were seeded at a density of 3 × 10⁵ cells per well in 6-well plates containing DMEM or RPMI 1640 medium supplemented with 10% FBS. Triplicate wells were prepared for all three independent experiments. Cells were then treated with 1 μM DAC and/or 1 μM BIX on the next day. After 12-h treatment, cells were digested with 0.25% trypsin, washed once with PBS, and resuspended in serum-free DMEM or RPMI 1640 medium. Subsequently, about 100 μL of the cell suspension (1 × 10⁵ cells) was added to the upper chamber of a transwell insert with 6.5 mm diameter and 8.0 µm pore size polycarbonate membrane (3422, Corning, New York, USA). The lower chamber was filled with 0.6 mL of culture medium containing 10% FBS. After incubating for 48 h, the top of the membrane was wiped with a cotton swab to remove non-migrated cells. Migrated cells on the bottom of the membrane were then fixed with 4% paraformaldehyde for 20 min and stained with 0.2% crystal violet for an additional 20 min. Finally, representative images were captured using an inverted optical microscope (Sunny Optical Technology). Transmigrated cells were manually counted using ImageJ software in randomly selected fields of view, and results were expressed as cell counts per field. At least three random fields were analyzed per group, and all experiments were independently repeated three times.
Transwell invasion assay
The procedure for the transwell invasion assay was similar to that of the transwell migration assay, with the exception that the membrane of the transwell insert was pre-coated with 1 mg/mL Matrigel matrix (356234, Corning) for 3 h at 37 °C. Subsequent steps involving cell seeding, incubation, fixation, staining, and quantification were identical to those in the transwell migration assay.
Apoptosis assay
TNBC cells were seeded at a density of 2 × 10⁵ cells in 12-well plates containing DMEM or 1640 medium supplemented with 10% FBS. Triplicate wells were prepared for subsequent experiments. After 24 h, cells were treated with 2 μM DAC and/or 2 μM BIX. Following a 48-h treatment period, cells were subjected to apoptosis detection using the Annexin V-FITC/PI apoptosis kit (AP101-30, LianKe Biotech, Hangzhou, China) according to the manufacturer’s instructions. Briefly, cells were resuspended in 500 μL of 1× Binding Buffer containing 5 μL of Annexin V-FITC and 10 μL of PI, followed by incubation in the dark for 5 min. Subsequently, quantitative analysis of Annexin V-positive and/or PI-positive cells was performed using a flow cytometer (CytoFLEX S, Beckman Coulter, California, USA) with detection parameters set at 488 nm and 561 nm lasers. The experiment was independently repeated three times. The total apoptotic rate was calculated as the sum of the proportions of Annexin V single-positive (early apoptosis) and Annexin V/PI double-positive (later apoptosis) cells. Simultaneously, cleaved caspase-3 was detected by western blot with antibodies against caspase-3 (14220 T, CST, Massachusetts, USA) or cleaved caspase-3 (9664 T, CST, Massachusetts, USA). Anti-GAPDH (60004-1-Ig, Proteintech, Tianjin, China) was used as a loading control. All samples were taken from the same batch of cells and subjected to identical treatment. DNMT1 WT and KO cells were treated for 48 h with the apoptotic inducers LCL161 (0.1 μM) and Cisplatin (50 μM). Apoptotic cell populations were quantified by flow cytometry. In parallel, levels of cleaved caspase-3 were assessed by western blot analysis. To further validate the role of DNMT1, DNMT1 was re-expressed in KO cells via transient transfection (rescue experiment). The rescued DNMT1-expressing KO cells were then treated in parallel with LCL161 (0.1 μM) or cisplatin (50 μM) for 48 h, and apoptotic cell populations were quantified by flow cytometry.
Lipid ROS analysis
TNBC cells were seeded at a density of 3 × 10⁵ cells in 6-well plates containing DMEM or 1640 medium supplemented with 10% FBS. Cells were treated in triplicate with 1 μM DAC and/or 1 μM BIX after 24 h. Erastin (1 μM) was used as a positive control. Another 48 h later, intracellular lipid reactive oxygen species (ROS) levels were assessed with 2 μM C11-BODIPY dye (D3861, Invitrogen) for 30 min in the dark, followed by washing with PBS 3 times. Cells were then resuspended in 200 μL of PBS. The oxidation of the polyunsaturated butadienyl moiety of the dye caused a shift in the fluorescence emission from approximately 590 nm (PE-positive) to about 510 nm (FITC-positive). The percentage of FITC-positive cells was quantified to represent lipid ROS levels. The mean fluorescence intensity (MFI) of FITC-positive cells was also measured. Additionally, TNBC cells were treated with 1 μM DAC and/or BIX for 48 h in the presence or absence of Ferrostatin-1 (Fer-1, 10 μM), and relative lipid ROS levels were assessed using the C11-BODIPY dye. For genetic validation, DNMT1 WT and KO cells were treated with erastin (1 μM) for 48 h, and lipid ROS levels were measured as described above. For rescue experiments, DNMT1 was re-expressed in KO cells via transient transfection prior to identical erastin treatment and analysis. Three independent experiments were conducted.
FerroOrange assay
TNBC cells were seeded in confocal dishes at a density of 3 × 10⁵ cells per dish. After 24-h attachment, cells were treated in triplicate with 1 μM DAC and/or 1 μM BIX. Subsequently, following an additional 48-h incubation, intracellular labile ferrous iron (Fe2+) levels were detected using BioTracker FerroOrange Live Cell Dye (SCT210, Sigma-Aldrich) according to the manufacturer’s instructions. Specifically, cells were incubated with 1 μM FerroOrange working solution in serum-free medium at 37 °C for 30 min in the dark. After washing with PBS, fluorescence images were immediately captured using a confocal fluorescence microscope (Ex/Em = 548/561 nm), and fluorescence intensity was quantified using ImageJ software. The fluorescence intensity of FerroOrange was regarded as an indicator of intracellular labile Fe2+ levels. The fluorescence intensity in the control group was normalized to 1 for relative quantification.
RSL3 inhibition assay
To evaluate the dose-dependent response of TNBC cells to RSL3, cells were seeded at 10,000 cells per well in 96-well plates, with three technical replicates per group and three independent biological replicates. After attachment, cells were cultured at 37 °C in a humidified incubator with 5% CO2 for 24 h. The medium was then replaced with fresh culture medium containing different concentrations of RSL3, and an equivalent volume of DMSO was added to the control group. After an additional 48 h of incubation, 10 μL of CCK-8 reagent was added to each well, followed by incubation at 37 °C for 4 h. Absorbance was measured at 450 nm. Cell viability and IC50 values were analyzed using GraphPad Prism 9 software.
Lipid ROS imaging
4T1, MDA-MB-231, and MDA-MB-468 cells were seeded in confocal dishes (D35-20-1-N, Cellvis, California, USA) at a density of 3 × 10⁵ cells per dish, in triplicate. Cells were treated with 1 μM DAC at 24 h post-seeding, while DMSO was used as the vehicle control. After 48-h treatment, cells were stained with C11-BODIPY and Hoechst (62249, ThermoFisher, Massachusetts, USA) dyes for 30 min at 37 °C and 5% CO2. Subsequently, these cells were washed with PBS and imaged utilizing a confocal microscope (LSM 980, Zeiss, Oberkochen, Germany). The intensity of the FITC-positive component indicated lipid ROS levels. Three independent imaging assays were performed.
DNMT1 knockout cell line construction
The sgRNA expression vector was generated by cloning the target sequence (sgDNMT1: 5’-TCCTCCCAGGCTCAAAGATT-3’) into the lentiCRISPRv2.0 vector (52961, Addgene, Massachusetts, USA). Pseudotyped viruses were produced by co-transfecting HEK293T cells with VSV-G glycoprotein-expressing vector pMD2.G (12259, Addgene), lentiviral packaging construct psPAX2 (12260, Addgene), and sgRNA-expressing construct lentiCRISPRv2.0. Subsequently, MDA-MB-231 cells were infected with the sgDNMT1 lentiviruses, followed by treatment with 1 μg/mL puromycin (A1113803, Gibco) to eliminate uninfected cells at 48 h post-infection. Three days post-puromycin selection, surviving cells were transferred to fresh DMEM medium for further cultivation. The efficiency of DNMT1 knockout was verified by western blot analysis using a rabbit polyclonal antibody against DNMT1 (5032 T, CST).
RNA extraction and sequencing
Total RNAs were extracted from wild-type (DNMT1-WT) and DNMT1-knockout (DNMT1-KO) MDA-MB-231 cells using TRIzol reagent (15596018, Invitrogen, California, USA). RNA samples from each cell type were prepared in duplicate to construct cDNA libraries. RNA purity and quantification were evaluated using a NanoDrop 2000 spectrophotometer (ThermoFisher), while RNA integrity was assessed using an Agilent 4200 Bioanalyzer (Agilent Technologies, California, USA). The RNA libraries required for RNA-sequencing (RNA-Seq) were prepared as follows. Poly(A)-enriched mRNAs were purified from total RNAs and fragmented into 300 to 350 bp pieces. The fragmented RNAs were reverse transcribed into cDNAs, followed by end repair, adenylation, and adapter ligation. The cDNA templates underwent PCR amplification, followed by purification of PCR products to obtain final libraries. The constructed RNA libraries were sequenced on the Illumina NovaSeq 6000 sequencing system by Berry Genomics. RNA-seq data have been deposited in the NCBI database with the following accession numbers: B231-WT-1 (SRR31392303), B231-WT-2 (SRR31392302), B231-KO-1 (SRR31392301), B231-KO-2 (SRR31392300).
For reverse transcription-quantitative polymerase chain reaction (RT-qPCR), total RNA from each cell type was extracted using an EZ-press RNA Purification Kit (B004D, EZBioscience, Minnesota, USA) according to the manufacturer’s instructions. cDNAs were synthesized from total RNAs via StarScript Pro All-in-one RT Mix Kit (A240-10, GenStar, Beijing, China), followed by qPCR with Taq Pro Universal SYBR qPCR Master Mix Kit (Q712-03, Vazyme, Nanjing, China) on a QuantStudio 1 Plus Real-Time PCR System (Applied Biosystems, California, USA) in triplicate. The relative expression levels of each gene were calculated by first normalizing to internal control genes, followed by normalizing to the control groups. Primers used for RT-qPCR in this study have been listed in Table S1 (Supplementary Material). We validated the DNMT1 knockout-induced changes in protein expression of c-Fos, GPX4, DAPK1, and KISS1 by western blotting. Western blot analyses were performed using antibodies against c-Fos (2250 T, CST), GPX4 (67763-1-Ig, Proteintech), DAPK1 (67815-1-Ig, Proteintech), and KISS1 (85068 T, CST), with GAPDH serving as the loading control.
SIM imaging
To prepare samples for super-resolution structured illumination microscopy (SIM) imaging, 4T1, MDA-MB-231, and MDA-MB-468 cells were seeded in a 6-well plate in triplicate with coverslips pre-treated with poly-L-lysine (polyK) (P1399, Sigma-Aldrich). These cells were transfected with GFP-tagged constructs at 24 h post-seeding. Another 24 h later, cells were fixed with 4% paraformaldehyde (16005, Sigma-Aldrich) and permeabilized with 0.2% Triton X-100 (A600198, Sangon Biotech, Shanghai, China). Subsequently, cells were incubated sequentially with specific primary antibodies and fluorescently labeled secondary antibodies. DNA was stained with 4’,6-diamidino-2-phenylindole (DAPI) (62248, ThermoFisher). For cells transfected with GFP-tagged constructs, only fixation, permeabilization, and DAPI staining were required. Coverslips with cells were mounted with ProLong Diamond Antifade Mountant (P36961, Invitrogen) on slides for imaging.
Cell samples were imaged using a Nikon A1 confocal microscope equipped with a CFI Apo TIRF objective (1.49 NA, oil immersion), an sCMOS camera (Hamamatsu Flash 4.0, pixel size 6.5 μm × 6.5 μm), four lasers (SIM 405, SIM 488, SIM 561, and SIM 647), and NIS-Elements AR software. During 3D-Stack SIM imaging, the z-axis step size was set to 0.20 μm per frame. For each focal plane, 15 images were captured in 3D-SIM mode (5 phases and 3 angles). The reconstruction and analysis of SIM images were performed using the N-SIM module of NIS-Elements AR software. The size, number, distribution, and sphericity of DNMT1 bodies were measured and analyzed using Imaris software (Imaris x64 9.0.1). Briefly, raw images were imported and background correction was applied to the FITC channel to remove noise while preserving target signals. The “Spots” function was used for detection, with the option “different spot sizes” enabled, and the FITC channel selected for analysis. The estimated spot diameter was set according to condensate size (approximately 0.1 μm for endogenous condensates and 0.2 μm for exogenous condensates). After automatic initial thresholding, manual adjustment was performed to ensure accurate identification of all discernible condensates while minimizing background signals. Parameters including spot number and diameter were exported for quantitative analysis of condensates.
FRAP experiment
The internal diffusion characteristics of DNMT1 bodies were analyzed through fluorescence recovery after photobleaching (FRAP) experiments. GFP-tagged DNMT1 constructs were transfected into MDA-MB-468 or HEK293T cells. Live cells were then imaged on a Zeiss Axio Observer Z1 confocal microscope equipped with a 63×/1.40 NA oil immersion objective. GFP fluorescence was excited using a 488 nm laser, with the pinhole set to 1.0 AU and signals detected using a GaAsP-PMT detector. Three condensates were selected as regions of interest (ROIs) for photobleaching (approximately 0.5 μm in diameter), which was performed using a 488 nm laser at 90% power with 100 scanning iterations. For HEK293T cells, image acquisition was conducted at 0.2% laser power with a time interval of 1.26 s per frame. For MDA-MB-468 cells, image acquisition was performed at 0.5% laser power with a time interval of 1.89 s per frame. Another three ROIs served as the unbleached negative controls. A total of 45 frames were acquired for each experiment. FRAP data were analyzed using ZEN 3.9 software (Zeiss), with background subtraction, normalization, and correction for overall photobleaching. Recovery curves were fitted using a single-exponential model, and the half-time of recovery (T1/2) was calculated. The FRAP assays were performed for three independent experiments.
Protein purification
The GFP-tagged DNMT1-DS1 and DNMT1-DS1 mutants were cloned into the pET28a vector containing a 6×His tag. The constructed clones were transformed into BL21 competent cells and cultured in LB medium containing kanamycin until the OD600 reached 0.4 to 0.6. The isopropyl β-D-1-thiogalactopyranoside (IPTG) (A600168, Sangon Biotech) solution was added to induce the expression of target genes for 18 h. Subsequently, the bacterial pellet was collected by centrifugation, followed by resuspension in Buffer A (50 mM Tris-HCl, pH 7.5, 500 mM NaCl). The bacteria were then lysed by sonication in an ice bath via VCX-500 Ultrasonic Microprocessor (Sonics Materials, Connecticut, USA) (20 min, 70% power, 5 sec on / 5 sec off). Cell debris and insoluble materials were removed by centrifugation. The cleared supernatant was incubated with Ni Sepharose Excel beads (17371202, Cytiva, New York, USA) to enrich the 6×His-tagged target proteins. The protein-bound beads were then washed sequentially with 30 mL of Buffer B (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 10 mM imidazole), Buffer C (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 20 mM imidazole), and Buffer D (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 50 mM imidazole). Target proteins were eluted stepwise using Buffer E (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 100 mM imidazole), Buffer F (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 200 mM imidazole), and Buffer G (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 500 mM imidazole). Elutions from Buffer E to G were concentrated and buffer-replaced with Buffer A using a 30 kDa ultrafiltration device.
In vitro droplet formation assay
Protein solutions were diluted into buffers with varying NaCl concentrations. The NaCl concentration gradients were prepared by serially diluting protein solutions with Buffer H (50 mM Tris-HCl, pH 7.5, 10% glycerol, 1 mM DTT). The droplet formation buffers included Buffer I (50 mM Tris-HCl, pH 7.5, 250 mM NaCl, 10% glycerol, 1 mM DTT), Buffer J (50 mM Tris-HCl, pH 7.5, 125 mM NaCl, 10% glycerol, 1 mM DTT), Buffer K (50 mM Tris-HCl, pH 7.5, 62.5 mM NaCl, 10% glycerol, 1 mM DTT), Buffer L (50 mM Tris-HCl, pH 7.5, 31.25 mM NaCl, 10% glycerol, 1 mM DTT), and Buffer M (50 mM Tris-HCl, pH 7.5, 15.625 mM NaCl, 10% glycerol, 1 mM DTT). Under different conditions of NaCl and protein concentrations, droplets were rapidly imaged using a Nikon A1 confocal microscope with a 488 nm laser. The areas of droplets in three preparations were calculated via Imaris software (Imaris x64 9.0.1) and analyzed by GraphPad Prism 9.
Molecular docking
For the binding mode of DAC to the catalytic domain of DNMT1, the crystal structure of human DNMT1 (351-1600) (PDB ID: 4WXX) was used as the receptor. DAC was docked to the catalytic pocket of DNMT1 using Schrödinger Maestro software in covalent docking mode with default parameters. For the binding mode of DAC to DS1 region of DNMT1, the structure of DS1 was predicted by AlphaFold 3 (AF3). The model with the highest-ranking score of 0.68 from the prediction results was chosen as the receptor model for subsequent molecular docking. The ranking score was calculated based on the composite score of normalized pLDDT, pTM, ipTM, fraction disordered, and clash score, according to the equation of 0.8×ipTM + 0.2×pTM + 0.5×disorder – 100×has_clash. The pTM value of the selected model was 0.28, while the value of fraction disordered was 0.8, and the clash score was 0.0. The ipTM provided the score of the predicted interface, which was not applicable to DS1 monomer prediction. The weights of both ipTM and pTM were assigned to pTM alone. The highest ranking score of 0.68 was calculated as follows: 1×0.28 + 0.5×0.8-100×0.0 = 0.68. DAC was docked to DS1 using AutoDock 4.2 software in blind docking mode. The model generated by AF3 only contains the protein backbone, without any heteroatoms or water molecules. Therefore, during docking preparation, no water molecules were added to either the DS1 receptor model or the DAC ligand model. Eight clusters with binding energies below -3.00 kcal/mol were the possible binding sites and selected for further analysis. The docking results were analyzed and visualized via PyMOL 3.1 software.
SPR assay
Surface plasmon resonance (SPR) measurements of the binding of DAC to recombinant GFP-tagged DNMT1-DS1 were performed using a Biacore 8 K+ instrument (GE Healthcare). A Biacore CM7 sensor chip and an amine coupling kit were employed according to the manufacturer’s protocol. The optimal pH for the immobilization of DS1 was determined to be 5.5. The surface of CM7 sensor chip was activated, followed by covalent coupling of DS1 (20 μg/mL in 10 mM acetate buffer, pH 5.5) for 10 min. Unreacted active esters were blocked by injecting 1 M ethanolamine-HCl for 7 min. All immobilization and binding analyses were conducted at 25 °C using PBS-P running buffer (10 mM phosphate, 137 mM NaCl, 2.7 mM KCl, and 0.05% P20, pH 7.4) supplemented with 5% DMSO. DAC solutions were prepared as a two-fold serial dilution series (ranging from 1.56 to 25 μM) in running buffer containing 5% DMSO. To minimize bulk refractive index effects, solvent correction was applied. A reference flow cell without immobilized DS1 was employed to account for non-specific binding. DAC at increasing concentrations was injected over the DS1-coated surface at a flow rate of 30 μL/min. The association phase was monitored for 120 s, followed by a 300-s dissociation phase in running buffer for each cycle. Response units were recorded at the end of the injection phase. Sensorgrams were fitted using the “Affinity” model implemented in the Biacore 8 K+ evaluation software to obtain binding parameters.
MST assay
Microscale thermophoresis (MST) assays were carried out using a Monolith NT.115 Pico instrument (NanoTemper Technologies) following established protocols. All measurements were performed in an assay buffer containing 50 mM Tris, 500 mM NaCl, and 0.02% (v/v) Tween-20, pH 7.5. To assess the binding affinity between DAC and GFP-tagged DNMT1-DS1, DAC ligand solutions were prepared through serial 2-fold dilutions in quadruplicate, with concentrations ranging from 1.5 nM to 50 μM. Each ligand dilution was incubated with a fixed concentration of GFP-DNMT1-DS1 (150 nM) at room temperature for 10 min. Following incubation, the samples were loaded into Monolith NT.115 Standard Treated Capillaries (MO-K002, NanoTemper Technologies), and MST measurements were performed using 20% LED power and medium MST power settings. All experiments were independently repeated with separate protein preparations to ensure reproducibility. Dissociation constants (KD) were determined based on the law of mass action using NanoTemper MO. Affinity Analysis (NanoTemper Technologies).
In vivo animal model
To establish an in vivo 4T1 TNBC lung metastasis model, 5-week-old female BALB/cJGpt mice (N000020, GemPharmatech, California, USA) were housed at Guangzhou National Laboratory Animal Center under specific pathogen-free (SPF) conditions. A saline suspension containing 1 × 10⁶ 4T1-luc cells (approximately 100 μL) was injected into BALB/c mice via the tail vein. Mice were randomly allocated to each group (n = 4 per group) with similar initial average body weights. Subsequently, mice were intraperitoneally injected with saline, DAC (2 mg/kg), BIX (2 mg/kg), and the combination of two compounds (2 mg/kg DAC + 2 mg/kg BIX), respectively, five times per week for two weeks. The body weight of each mouse was monitored every two days. Bioluminescence imaging was conducted weekly to monitor lung metastasis. The bioluminescence quantification was performed based on total photon flux. All mice were euthanized after two weeks. The metastatic nodule quantification was conducted based on manual microscopic examination of lung tissues by two investigators.
For the in vivo MDA-MB-231 TNBC orthotopic tumor model, approximately 1 × 10⁶ MDA-MB-231 cells were orthotopically implanted into the mammary fat pad of 5-week-old female immunodeficient NCG mice (NOD/ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22/Gpt) (T001475, GemPharmatech). One week after inoculation, mice were randomly assigned to four groups (n = 4 per group) and intraperitoneally injected with saline, DAC (2 mg/kg), BIX (2 mg/kg), and the combination of two compounds (2 mg/kg DAC + 2 mg/kg BIX), respectively, twice a week for three weeks. Tumor volume was measured three times per week using calipers and calculated as (length × width²)/2. Body weight was recorded every two days. Mice were euthanized on day 25 and tumors were harvested.
For the in vivo MDA-MB-231-luc TNBC lung metastasis model, the procedure was identical to that in the 4T1 lung metastasis model, except that compound treatments began on day 7 post-tumor inoculation in NCG mice (n = 4 per group), continuing for two weeks with two doses per week. Bioluminescence imaging was conducted twice a week to evaluate therapeutic effects. All animal experiments were approved by the Ethics Committees of the Guangzhou Medical University Animal Center and the Guangzhou National Laboratory Animal Center.
Ki67 immunostaining of mouse lung or tumor tissues was quantitatively analyzed using ImageJ software. The IHC images were transformed into 8-bit grayscales, and a consistent threshold was applied to identify positive signals. The integrated density (IntDen) was measured, and the IntDen/Area ratio was calculated using the entire image area serving as the normalization baseline to represent Ki67 expression. The control group was set to a value of 1, and the relative Ki67 expression levels of the experimental groups were calculated accordingly. Statistical analysis was performed by comparing the relative values among groups.
Statistical analysis
Statistical analysis was performed using GraphPad Prism 9 or Microsoft Excel. Comparisons between two groups of data were conducted using Student’s t-tests, while comparisons between multiple groups were performed utilizing one-way or two-way ANOVA. All results were presented as mean ± SD. Network analysis and clustering analysis were performed using the STRING database. Statistical significance was defined as follows: P values < 0.05 (*), < 0.01 (**), < 0.001 (***), and < 0.0001 (****).
