Human 293t cells

Human gastric cancer cell lines (AGS, N87) were purchased from the American Type Culture Collection (ATCC), MNK-45 and SGC-7901 were obtained from the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ). Human normal gastric cell line (GES-1) and human 293T cells were purchased from ATCC. Cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM; Sigma-Aldrich, USA), plus 10% fetal bovine serum (FBS; Gibco, USA). Cells were maintained at 37 ℃ with 5% CO₂. Human gastric cancer and corresponding paracancerous tissues were obtained from Hepatobiliary Pancreatogastric Surgery, Shanxi Cancer Hospital in February 2022. Tissues were immediately soaked into RNA later (beyotime, China) after excision. All tissues were saved at -80 ℃ until used. Following criteria were excluded: (1) patients younger than 18 years, older than 75 years, and without full civil capacity; (2) patients who were diagnosed as cancer in other organs; (3) patients received anti-cancer therapies before surgery. This study was approved by the ethics committees of Cancer Center of Shanxi province. Informed consents were obtained from all participants.

Human 293T cells (ATCC, LGC Standards, Molsheim, France) were transfected with the indicated plasmids using Lipofectamine 2000 (Invitrogen), according to the manufacturer’s protocol. At 15 h post-transfection, cells were harvested and lysed in non-denaturing lysis buffer (10 mM Hepes, 5 mM EDTA, 150 mM NaCl, 1% NP40, and 50 µM PR-619 (662141, Calbiochem; Merck, Darmstadt, Germany) supplemented with protease inhibitors (11873580001, Sigma-Aldrich). Lysates were clarified for 10 min at 10,000 rpm. Supernatants were then incubated with M2 anti-FLAG magnetic beads (M8823, Sigma-Aldrich) for 2 to 3 h at 4 °C under agitation. The beads were washed four times in lysis buffer before boiling 15 min in loading buffer. For Western blots, equal amounts of protein were loaded and separated on 4–15% gradient precast gels and transferred onto PVDF membranes before staining. Samples were immunoblotted with the appropriate primary antibodies. Protein levels were quantified by measuring the intensity of the bands by densitometry. Actin or GAPDH were used as positive controls of the cell extracts.

Human 293T cells (ATCC) were cultured in DMEM containing 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin and 4 mM L-glutamine. Mouse multipotent HSC/MPP-like cell line EML cells (ATCC) were cultured in IMDM supplemented with 4 mM L-glutamine, 100 ng/ml mSCF, 20% FBS, 100 µg/ml streptomycin and 100 U/ml penicillin.

Gene-editing experiments were performed in a single parallel culture experiment using human 293T cells (ATCC) grown in DMEM with 10% FBS. On day zero, 500,000 cells were seeded per well in a six-well plate format. The following day, two master mixes were prepared: a) LT-1 transfection reagent (Mirus) was diluted 1:10 in Opti-MEM and incubated for five minutes; b) a DNA mix of 500 ng Cas9-sgRNA plasmid with 250 pmol repair template oligonucleotide (Supplemental Information) was diluted in Opti-MEM in a final volume of 100 μL. 100 μL of the lipid mix was added to each of the DNA mixes and incubated at room temperature for 25 minutes. Following incubation, the full 200 μL volume of DNA and lipid mix was added drop-wise to the cells, and the cells were centrifuged at 1000xg for 30 min. Six hours post-transfection, the media on the cells was replaced, and the cells were passaged as needed. On day six, five million cells from each condition were pelleted to serve as an early time point for the editing efficiency, and five million cells were then passaged on the five drugs at two doses for 12 days, at which time all surviving cells were pelleted. Concentrations used for each small molecule are: etoposide– 500 nM, 100 nM; amasacrine– 500 nM, 100 nM; teniposide– 20 nM, 4 nM; dactinomycin– 4 nM, 800 pM; and XK469–5 μM, 1 μM.

HepAD38 is an inducible hepatoblastoma cell line that contains an HBV infectious clone under the control of a tetracycline (tet)-off regulated cytomegalovirus (CMV) promoter; HBV is induced when tet is removed from the culture medium [31 (link)]. HepG2 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). HepG2-NTCP clone 3E8 and HepG2-NTCP clone 7 cells were gifts from Drs. Charles Rice and Eleftherios Michailidis (Rockefeller University, NY, USA). HepG2-NTCP clone 3E8 and HepG2-NTCP clone 7 cells were described previously [33 (link),34 (link)]. Human 293T cells were from the American Type Culture Collection. In general, all cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS). For HepAD38 cells, 0.3 µg/mL tet and 400 µg/mL G418 were added to the culture medium, as previously described [31 (link)]. Cells were incubated at 37 °C in the presence of 5% CO₂.