Rpmi 1640 medium

HeyA8 (RRID:CVCL_8878) and HeyA8 CD95 knock-out cells, HCT116 (ATCC #CCL-247; RRID:CVCL_0291) and HCT116 Drosha knock-out and Dicer knock-out cells, MCF-7 cells (ATCC #HTB-22; RRID:CVCL_0031), and 293T (ATCC #CRL-3216; RRID:CVCL_0063) cells were cultured as described previously (Putzbach et al., 2017 (link)). The MCF-7 CD95 knock-out and deletion cells were cultured in RPMI 1640 medium (Cellgro #10–040 CM), 10% heat-inactivated FBS (Sigma-Aldrich), 1% L-glutamine (Mediatech Inc), and 1% penicillin/streptomycin (Mediatech Inc). H460 (ATCC #HTB-177; RRID:CVCL_0459) cells were cultured in RPMI1640 medium (Cellgro Cat#10–040) supplemented with 10% FBS (Sigma Cat#14009C) and 1% L-glutamine (Corning Cat#25–005). 3LL cells (ATCC #CRL-1642; RRID:CVCL_4358) were cultured in DMEM medium (Gibco Cat#12430054) supplemented with 10% FBS and 1% L-glutamine. Mouse hepatocellular carcinoma cells M565 cells were described previously (Ceppi et al., 2014 (link)) and cultured in DMEM/F12 (Gibco Cat#11330) supplemented with 10% FBS, 1% L-glutamine and ITS (Corning #25–800-CR). All cell lines were authenticated using STR profiling and tested monthly for mycoplasm using PlasmoTest (Invitrogen).

The Nalm6 cell line is previously described46 (link). The CCRF-CEM (CEM), MOLT-4 and U-937 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured in RPMI-1640 medium (Cellgro, Tewksbury, MA, USA) supplemented with 10% fetal bovine serum (Hyclone, Logon, Utah, USA). HEK 293T cells were cultured in DMEM (Cellgro) supplemented with 10% fetal calf serum and 1% L-glutamine (Cellgro). Cells were incubated at 37 °C in a humidified atmosphere with 5% CO₂. Primary human B- and T-cell ALL cells were cultured in RPMI–1640 medium (Cellgro) supplemented with 10% fetal bovine serum (GE-Hyclone, Logon, Utah, USA). 4,5,6,7-Tetrabromobenzotriazole (TBB) and CX-4945 were purchased from Sigma (St. Louis, MO, USA). Cells were cultured with or without TBB and collected for total RNA isolation. Human IKZF1 retroviral construct and retroviral production was described15 (link)17 (link)47 (link)48 (link).

Cells were incubated at 37°C in a humidified atmosphere of 5% CO_2. Nalm6 cells have been previously described [27 (link)]. CCRF-CEM (CEM), MOLT-4 and U-937 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cell lines werecultured in RPMI 1640 medium (Cellgro) supplemented with 10% fetal bovine serum (Hyclone). HEK 293T cells were cultured in DMEM (Cellgro) supplemented with 10% fetal calf serum and 1% L-glutamine (Cellgro). Primary human B-ALL cells were cultured in RPMI 1640 medium (Cellgro) supplemented with 10% fetal bovine serum (Hyclone). Cells were cultured with or without TBB and collected for total RNA isolation. Human HA-tagged Ikaros (IKZF1) retroviral construct and retroviral production was described previously [27 (link)–30 (link)].

YTN5 and YTN16 cells were transfected with FOXM1 siRNA for 24 h and then exposed to 20 ng/mL IFNγ (Abcam, #9922) for 24 h. YTN5 and YTN16 cells were then trypsinized (Gibco, #15400-054) and seeded at 1 × 10⁵ in a 24-well plate and then pulsed with 1 ng/mL OVA peptide (Sigma, #S7951-01mg) for 3 h. Simultaneously, spleens were removed from C57BL/6-Tg (TcraTcrb) mice and splenocytes were treated with 1X Red Blood Cell lysis buffer (eBioscience, #00-4333-57) for 5 min. CD8⁺ T Cells were isolated using the MojoSort™ Mouse CD8⁺ T Cell Isolation Kit (Biolegend, #480035) and resuspended in RPMI-1640 medium (Corning, #45000-396) containing 20% FBS (Omega Scientific, #FB-02). CD8⁺ T cells were incubated with 100 U/mL IL2 (Abcam, #ab9856) for 24 h, followed by 300 ng/mL OVA peptide for 24 h. CD8⁺ T cells were spun down at 1500 × g for 5 min and resuspended at 4 × 10⁵ cells/100 μL of RPMI-1640 medium (Corning, #45000-396) containing 20% FBS (Omega Scientific, #FB-02). Media from YTN5 and YTN16 cells was removed and replaced with 400 μL of fresh culture media, followed by 100 μL of CD8⁺ T cell containing media. The co-cultured cells were incubated for 24 h. The media and suspended CD8⁺ T cells were removed, and alive cancer cells were stained with 1% crystal violet as described above.