Dmem medium

The human breast cancer cell lines (MCF-7, ZR-75-1, and MDA-MB-231) and HEK293T-17 were obtained from the American Type Culture Collection (Maryland, USA). SUM159PT cells were obtained from Asterand Bioscience (Detroit, Michigan, USA). MCF-7 and HEK293T-17 cells were cultured in DMEM medium (Gibco, California, USA) with 5% FBS (ExCell Bio, Suzhou, China), supplemented with 1% penicillin/streptomycin (Beyotime Biotechnology, Jiangsu, China). ZR-75-1 cells were were cultured in DMEM with 10% FBS, supplemented with 1% penicillin/streptomycin. MDA-MB-231 cells were cultured in MEM medium (Gibco) containing 10% FBS, 1% penicillin/streptomycin, and 1.8 μg mL⁻¹ insulin (Solarbio, Beijing, China). SUM159PT cells were cultured in DMEM/F-12 medium with 5% FBS, 1% penicillin/streptomycin, 5 μg mL⁻¹ insulin, 1 μg mL⁻¹ hydrocortisone. MCF-7, ZR-75-1, and HEK293T-17 cells were incubated in tissue culture incubators with an atmosphere of 7.5% CO₂ at 37 °C, while MDA-MB-231 and SUM159PT cells were cultured with an atmosphere of 5% CO₂. All cell lines were characterized by DNA fingerprinting and isozyme detection.
MG-132 was purchased from Selleck (Texas, USA). Cycloheximide was purchased from Sigma (Darmstadt, Germany).

KYSE150, KYSE510, KYSE140, KYSE180, KYSE70, TE1, TE3 and NCM460 cells were maintained in RPMI-1640 medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (GIBCO), 100 U/mL penicillin G, and streptomycin. DMEM medium (GIBCO) supplemented with 10% fetal bovine serum was used for HEK293T, HeLa and FHC cell culture. HUVEC cells were grown in a fully supplemented Endothelial Cell Medium (ScienCell). ARPE-19 cells were cultured with DMEM-F12 containing 10% fetal bovine serum. ESCC cell lines were established and provided by Dr. Shimada Yutaka (Faculty of Medicine, Kyoto University, Japan) and Dr. Nishihira (Tohoku University School of Medicine, Japan)^{61 (link)–63 (link)}. NCM460 cells were established and provided by Dr. M.P. Moyer (University of Texas, San Antonio, TX)^{64 (link)}. ARPE-19 (GNHu45) and HeLa (SCSP-504) cells were obtained from National Collection of Authenticated Cell Cultures (Shanghai, China). HEK293T (CRL-3216) and FHC (CRL-1831) cells were obtained from American Type Culture Collection (ATCC). Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂. Cell lines were authenticated by short tandem repeat profiling and were routinely tested for mycoplasma contamination^{5 (link)}. For small interfering RNA (siRNA) knockdown, siRNAs targeting human ANLN or negative control siRNA were diluted in Opti-MEM reduced serum medium (Life Technologies, 31985070) and mixed with siRNA Transfection Reagent (Life Technologies, 13778150), according to the manufacturer’s instructions. Cells were harvested 24 or 48 h after transfection^{5 (link)}. Dox-induced ANLN-depleted and control cells were generated in KYSE150 cells as previously described^{65 (link)}. Short hairpin RNA (shRNA) targeting the 3’ UTR region of ANLN was constructed into a Dox-induced expression plasmid and stably expressed in KYSE150 cells by lentiviral infection. For rapid depletion of ANLN, cells were treated with 2 μg/mL Dox for 24 h. The sequences of siRNA and shRNA are listed in Supplementary Data 1. For 1,6-HD (Sigma, 240117) treatment, HeLa cells were cultured in 24 well plates containing glass coverslips, treated with 5% 1,6-HD for 60 s, immediately fixed with 4% paraformaldehyde and then used for immunofluorescence assay. For THZ1 (MedChemExpress, HY-80013) treatment, KYSE150 cells were cultured in 24 well plates, treated with different concentrations of THZ1 for 3 h, and observed with an inverted microscope to determine a low toxicity concentration, which was selected for immunofluorescence assay. For pre-extraction, cells were treated with cold cytoskeleton buffer (0.5% Triton X-100, 10 mM PIPES pH 6.8, 3 mM MgCl₂, 200 mM NaCl, 300 mM sucrose) for 5 min at 4 °C, followed by fixation with 4% paraformaldehyde, and the samples were used for immunofluorescence assay^{21 (link),22 (link)}.

Take the 0-day fertilized egg, wipe it clean with new disinfectant, and then disinfect it with 75 %. Place it on the clean bench and gently knock it open with tweezers. Take the embryonic disc, place it in the prepared pre-cooled PBS, wash it twice and put it in a 15 mL centrifuge tube. After collecting the embryonic disc, absorb the excess PBS solution, gently blow away the cells, centrifuge, discard the supernatant, use 3 mL of DMEM medium (Gibco) supplemented with 10 % fetal bovine serum (Gibco) and 1 % penicillin/streptomycin (Penn/Strep), gently resuspend, filter into a clean 15 mL centrifuge tube, transfer to a clean cell culture dish, and culture at a differential rate (Growing cells at different rates to study how different growth or division rates affect their differentiation into specific types) for 16 h. Collect the cell fluid in the dish into a 15 mL centrifuge tube, centrifuge it at 1400 r for 6 min, discard the supernatant, resuspend it in 1 mL DMEM medium, count, observe the cell morphology and take pictures.
After 6 days of incubation, the medium was discarded, and cells were washed twice with PBS and fixed with paraformaldehyde (Solarbio, P1110) for 5 min. The cells were then stained with ALP staining solution (Solarbio, G1480) to assess pluripotency, as ALP is a marker of undifferentiated stem cells. After 30 min of incubation at room temperature, the cells were washed and stained with nuclear solid red staining (Solarbio, Beijing, China, G1480) for 5 min to highlight the cell nuclei. Following another PBS wash, the cells were observed under a microscope, and images were captured. The ALP staining results provide insights into the pluripotency of stem cells, with stronger staining indicating a higher degree of undifferentiation. Bright-field microscopy was used to capture both cell morphology and staining results.

Human nasopharyngeal epithelial cells (NP-69, SNL-565) and NPC cells C666–1 (SNL-516), HK-1 (SNL-563) were supplied from Sunncell Biotechnology Co., Ltd (Wuhan, Hubei, China). NPC cell line HNE-3 (Cs-010-006177) was purchased from Tongwei Biotechnology Co.,Ltd (Beijing, China), and NPC43 (CVCL_UH64) were obtained from BioVector Science Lab,Inc. (Beijing, China). The above cells were grown in DMEM medium (11,965,092, Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (F0193, Sigma-Aldrich, St. Louis, MO, USA) and 1% penicillin/streptomycin mixture (15,140,122, Gibco). The fluid change interval was 3 days, the passaging ratio was 1:3. The temperature was set to 37 °C and placed in an environment with saturated humidity and 5% volume CO₂.
SATB2 overexpressing vector (OE-SATB2) and its control (OE-NC) were produced by Sangon Biotech (Shanghai, China). Referring to the instructions of Lipofectamine 3,000 (L3000001, Invitrogen, Austin, TX, USA), control and experimental vectors were transfected into NPC cells. After 48 h of transfection, the transfection efficiency was reflected by detecting the expression level of SATB2 in cells.
In the AS-IV cytotoxicity assay, the concentration of AS-IV (HY-N0431, MedChemExpress, Monmouth Junction, NJ, USA) was 50, 100, 200, 400, or 800 μM, and the treatment time was 24 h. Based on the findings of the toxicity assay, 100, 200 and 400 μM of AS-IV were subsequently selected to treat NPC cells. In the signaling axis probe experiment, the Control+OE-NC group was transfected with OE-NC in NPC cells. The AS-IV + OE-NC group was exposed to AS-IV (400 μM) for 24 h after transfection of OE-NC in NPC cells. The AS-IV + OE-SATB2 group was exposed to AS-IV (400 μM) in NPC cells for 24 h after transfection of OE-SATB2 in the NPC cells. For the AS-IV + OE-SATB2 + DKK1 group, after transfection of OE-SATB2 in NPC cells, the NPC cells were exposed to AS-IV (400 μM) for 4 h, and then exposed to Dickkopf-related protein 1 (Dkk-1, 100 ng/mL, HY-P72968, MedChemExpress), a Wnt pathway inhibitor, treatment for 24 h.

MCF-7 and T47D breast cancer cells were purchased from the American Type Culture Collection (ATCC). MCF-7 cells were grown in DMEM medium (Gibco, USA) with 10% FBS (Hyclone, USA), while T47D cells were cultured in RPMI-1640 medium (Gibco, USA) plus 10% FBS (Hyclone, USA). Palbociclib-resistant MCF-7 and T47D cells have been established at our laboratory previously [35 (link)] and were cultured in DMEM or RPMI-1640 medium with 10%FBS plus 6 μM palbociclib.

The BoLC.8M3 lung adenocarcinoma cell line was derived from the spontaneous lung tumor of a BALB/c transgenic mouse model in our laboratory. This transgenic model harbored the oncogenic KRAS^G12D mutation and a heterozygous knockout of the p53 gene.
The cell line was established and cultured in DMEM medium (Thermo Fisher Scientific, Monza, Italy) supplemented with 20% FBS, 100 U/mL penicillin and 10 µg/mL streptomycin (Thermo Fisher Scientific). Cells were cultured at 37°C in a humidified 5% CO₂ atmosphere and were split once or twice a week according to density.