Cell culture

Jurkat, 6 T-CEM, CCRF, as well as J.gamma1 were obtained from the Chinese Academy of Sciences Cell Bank.SUP-T1and PF-382 were obtained from Zhejiang Meisen Cell Technology Co.,Ltd. The aforementioned cell lines were propagated in RPMI 1640 medium(22,400,089, Gibco, USA) supplemented with 10% fetal bovine serum(FBS)(Biological Industries, CT, USA). HEK293FT cells were propagated in full-strength DMEM medium (manufactured by Biological Industries; Sartorius AG), enriched with 10% FBS. Cells were cultured in a humidified incubator at 37℃ containing 5% CO2, and routinely tested for mycoplasma. Each cell line was authenticated through Short Tandem Repeat (STR) analysis.

Lentiviral preparation and shRNA-mediated knockdown (KD) of LDB1

The shRNA was synthesized and its incorporation into the pLKO.1 vector were carried out by IGE Biotechnology LTD, China. HEK293FT cells of 10 cm dish size were transfected with 7.5 μg of purified plasmids together with packaging plasmids, 5.625 μg of and psPAX2 and1.875 μg of pMD2.G(MA,USA), Using PEI transfection reagent. The supernatant was harvested after incubating for 48 h at 37℃, subsequently filtered using a 0.45 μm-syringe filter, and then applied for infecting cells that were plated at a density of 2 × 105 cells per well in a 6-well plate. After a 24-h incubation, the culture medium was refreshed with complete 1640 medium enriched with 1 µg/ml puromycin (ST551, Beyotime, China) to facilitate the selection of successfully infected cells. Three days following the selection process, we harvested the cells to achieve full knockdown of LDB1. Consequently, the infected cells were employed for various experiments. The sequences of shRNA utilized in this research are provided in Supplementary Table 1.

Reverse transcription–Real-time quantitative PCR

Using the prescribed manufacturer's instructions, the collected cells were subjected to RNA extraction utilizing the TRIzol® reagent (Invitrogen, CA, USA). Subsequently, the extracted total RNA was reverse transcribed into cDNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, CA, USA). For PCR amplification, the reaction system was prepared with LightCycler®480 SYBR Green I Master mixture (Roche, Germany), and real-time PCR was performed on the LightCycler 480 system (Roche). The CT values corresponding to the amplification cycles of both the experimental samples and controls were standardized to the levels of GAPDH. Subsequently, the comparative gene expression levels were determined through the calculation of fold changes utilizing the 2^–ΔΔCT method. The quantification of a gene's relative expression level was derived by taking the mean of three separate measurements. The sequences of primers utilized within the research are provided in Supplementary Table 2.

Western blotting analysis

Cells harvested underwent a cleansing step with phosphate-buffered saline and were subsequently subjected to lysis with RIPA buffer (Beyotime, China). Protein samples, quantitatively matched, underwent separation via SDS-PAGE, following which they were applied to polyvinylidene difluoride (PVDF) membranes (CST, USA). After blocked with a 5% non-fat milk solution in TBST and incubated with primary antibodies while being agitated at a temperature of 4℃ throughout the night, the PVDF membrane was incubated with a secondary antibody at room temperature. Then the membrane was coated with an ECL detection solution (Millipore, USA) to visualize the protein bands. The chemiluminescence emitted was captured with an AI600 gel documentation system (GE, USA) for image analysis. The antibodies employed in this research are documented in Supplementary Table 3.

CCK-8 and soft agar colony formation assay

Subsequent to puromycin-mediated selection, the infected cells were seeded at a concentration of 5,000 cells per well into 96-well plates. 20 μl of CCK-8 solution(APExBIO, K1018-1, USA) was dispensed to every individual well containing 200 μl of 1640 medium with infected cells on the 1st/3rd/5th days. The plates underwent incubation at a temperature of 37℃ for 2 h, post which a spectrophotometer (Thermo, USA) was utilized to measure the absorbance at a wavelength of 450 nm. Cells from both the shRNA negative control and shRNA targeting LDB1 groups were seeded into a soft agar culture medium. Following a period of approximately two weeks of incubation, the cells underwent a staining process using Giemsa stain (Product code C0131 from Beyotime, China), subsequently enabling the counting of cell colonies formed.

EdU assays

For 5-ethynyl-2-deoxyuridine Edu assays, the proliferation ability of cells was evaluated using a BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 594 (Cat#: C0078S; Beyotime).The procedure was performed according to the manufacturer’s instructions. Prepare control and LDB1 knockdown cells in advance. Subsequently, collect control and experimental group cells after 5 days of puromycin selection for EdU staining. After EdU staining, analyze the collected control and experimental group cell suspensions using flow cytometry.

Colony formation assay

Add 20 mL of Iscove's Modified Dulbecco's Medium (Catalog #36,150, Stem Cell Technologies, Canada) to 80 mL of MethoCult™ H4230(Catalog #04230, Stem Cell Technologies, Canada) methylcellulose medium and mix thoroughly by vigorously shaking the bottle for 1–2 min. Then, let the mixed MethoCult medium sit at room temperature for 15–20 min to allow bubbles to rise to the top. Aliquot the medium into sterile distribution tubes, with 3 mL in each tube, for later use. Two days after puromycin selection, both transfected control cells and LDB1 knockdown cells were collected. Dilute the cells from both control and experimental groups in IMDM medium containing 2% FBS to achieve a 10X final concentration. Add 0.3 mL of the cell suspension to each aliquoted 3 mL MethoCult medium and vortex for 5 min. Allow bubbles to rise to the top, then plate the cell suspension into 6-well plates at a final concentration of 10,000 cells/mL. To ensure adequate humidity, add sterile double-distilled water to the adjacent wells of the cell culture plates. Incubate the cells in a humidified incubator at 37 °C with 5% CO2. Colony formation was observed 10 days post-plating.

Cell apoptosis assay

The infected cells after puromycin selection were initially rinsed with chilled 1 × phosphate-buffered saline (PBS) first. Subsequently, the samples were resuspended in 1 × Annexin V binding buffer and labeled with FITC-Annexin V antibody as well as propidium iodide (PI) following the guidelines provided with the FITC-Annexin V Apoptosis Detection Kit (catalog no. 556547, BD Biosciences, USA). Apoptotic cells were quantified utilizing the Gallios™ Flow Cytometer from Beckman (Beckman Coulter, Krefeld, Germany).

Cell cycle assay

Following the manufacturer’s protocol, samples used for cell cycle assays were fixed overnight with pre-cooled 75% alcohol at 4 °C overnight. On the following day, the cells were washed with cold PBS and resuspended with PI dye and RNase A (cat. No. 550825; BD Pharmingen™, San Diego, CA, USA) and then incubated at room temperature for 15 min. The cell cycle was analyzed using Beckman Gallios™ flow cytometry (Beckman, Krefeld, Germany).

RNA‑seq analysis and data processing

RNA extraction, library preparation, transcriptome sequencing (on the Illumina NovaSeq 6000 platform), and raw data filtering were conducted by Novogene Bioinformatics Technology Co., Ltd. (Beijing, China). Alignment of the 150 bp paired-end sequences was executed against the hg38 (Ensembl) human reference genome using HISAT2 (version 2.2.0). The reconstruction and quantification of the transcriptome were carried out employing StringTie (version 2.1.2). The R/Bioconductor package DESeq2 facilitated the identification of genes exhibiting differential expression, adhering to a threshold of an adjusted P-value < 0.05 and an absolute fold change (FC) >| 1|. Gene Set Enrichment Analysis (GSEA) was carried out with the R/Bioconductor package clusterProfiler, leveraging the wikipathways collection (2023.1 release) sourced from the Molecular Signatures Database (MSigDB).

In vivo experiments

Female NSG mice aged 4–6 weeks were purchased from Shanghai Nanfang Model Biotechnology Inc. 6 T-CEM cells expressing firefly luciferase were prepared beforehand and transfected with viral particles carrying either sh-NC or sh-LDB1. Samples were collected on the third day following transfection. The NSG mice were divided into two groups using random assignment, with each mouse receiving an intravenous tail vein injection of a PBS-based cell suspension, consisting of 2 × 10^6 cells. D-luciferin sodium salt (GOLDBIO, USA) was administered intraperitoneally to tumor-bearing mice on D21/28/35 days after tail vein injection respectively. Then, using a small animal live imaging scanner from Berthold, Germany, quantification of the maximal standard mean radiance emitted by mouse tumors was performed. Liver, spleen, and bone tissues were harvested from both the experimental and control groups for the purposes of performing H&E staining and immunohistochemistry.

Cleavage under targets and tagmentation (CUT&Tag)

Assay

The CUT&Tag experiment was conducted on human 6 T-CEM, Molt4, Loucy and J.gamma1 cells utilizing the Hyperactive Universal CUT&Tag Assay Kit (TD903-01, Vazyme) following the guidelines provided by the manufacturer. 2 × 106 cells were incubated with cold Nuclear Extract Buffer for 10 min. After centrifugation, the cell precipitate was resuspended using 500 μl of a 0.1% formaldehyde solution in the sample tube. The sample was then incubated at room temperature for 2 min before the cross-linking reaction was Terminated by adding 2.5 M glycine. The cell precipitate obtained after centrifugation was collected and was resuspended using a 100 μl wash buffer. Subsequently activated ConA Beads were added for further incubation, and then mixed with primary antibodies, which include: LDB1, ERG, IRF1, ETS1, ETV6, and IgG mentioned above. The samples, along with the aboving antibodies, were incubated overnight at 4 °C. The following day, 50 μl of secondary antibodies diluted at a Proportion of 1:100 were added to each sample. The mixtures were then subjected to rotational incubation at room temperature for 1 h. The samples were next fragmented and DNA were extracted. Novogene carried out the sequencing of all CUT&Tag libraries, employing the PE150 protocol on the Illumina NovaSeq 6000 sequencing platform(Novogene, Beijing, China).

Immunoprecipitation

6 T-CEM and J.gamma1 cells (transfected with Ldb1-Flag overexpression constructs) were lysed using a buffer including NP-40 (P0013F, Beyotime, China) along with a protease inhibitor. Following a 30-min incubation at 4 °C in a refrigerated centrifuge, the lysate was centrifuged to obtain the supernatant for collection. Subsequently, A total of 50 μl lysate was mixed with 5 × loading buffer and boiled at 100℃ for 10 min to denature the proteins, serving as the input group. The protein A/G beads (B23202, Bimake, China) were mixed with the other two parts of the supernatant, while LDB1 antibody or IgG Antibodies were used in the respective mixtures. The mixtures were subsequently subjected to overnight rotation at 4 °C. The next day, the samples were subjected to centrifugation at 8200 g to remove the supernatant. Subsequently, the pellets were washed five times with pre-chilled TBST to remove the remaining supernatant. The pellets were then re-suspended in 2 × loading buffer and heated to induce denaturation, for subsequent use in Western blot analysis.

Statistical analysis

Three separate experiments were executed. All statistical analyses were performed using GraphPad PRISM 8.0.2 software. The two groups were compared by double-tailed unpaired Student's t test for data analysis. Statistical significance was ascribed to P values less than 0.05 as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Survival curve analysis was compared by Log-Rank test, and P < 0.05 indicate statistical significance.