Penicillin streptomycin

The patient-derived GSCs 84NS (RRID: CVCL_C6J0) and 528NS (RRID: CVCL_C6IV) were kindly provided by Dr. Ichiro Nakano (University of Alabama, Birmingham, AL, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 (GE Healthcare, Chicago, IL, USA) supplemented with 0.2% B27 (Invitrogen, Carlsbad, CA, USA), 20 ng/mL epidermal growth factor (EGF) (R&D Systems, Minneapolis, MN, USA), 20 ng/mL basic fibroblast growth factor (R&D Systems), 1% penicillin/streptomycin (GE Healthcare), 2 mmol/L L-glutamine (GE Healthcare), and 50 μg/mL gentamicin (Mediatech, Manassas, VA, USA). HEK293T (RRID: CVCL_0063), LN18 (RRID: CVCL_0392), A1207 (RRID: CVCL_8481), and A172 (RRID: CVCL_0131) cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). These cell lines were cultured in DMEM (GE Healthcare) supplemented with 10% fetal bovine serum (GE Healthcare), 1% penicillin/streptomycin, 2 mmol/L L-glutamine, and 50 μg/mL gentamicin. MG132, CQ, VER-155008, and CHX were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ganetespib was purchased from Abmole Bioscience (Houston, TX, USA). Cell lines used in this study were regularly monitored to ensure the mycoplasma-free cells. In addition, all human cell lines were authenticated in 2019 using short tandem repeat profiling.

A549, a cell line derived from primary lung cancer, and MRC-5, a human fibroblast cell line derived from normal lung tissue, were purchased from the American Type Culture Collection (ATCC, Rockville, MD). A549 cell line was cultured in F12K (ATCC) supplemented with 10% FBS (Euroclone, Milan, Italy), 1% penicillin/streptomycin (GE Healthcare, Milan, Italy) and 2% amphotericin B (Euroclone). MRC-5 cells were maintained in EMEM (ATCC) supplemented with 10% FBS (Euroclone), 1% penicillin/streptomycin (GE Healthcare) and 2% amphotericin B (Euroclone). All the cell lines were checked periodically for mycoplasma contamination using the MycoAlertTM Mycoplasma Detection Kit (Lonza, Basel, Switzerland). Before seeding in the bioreactor culture vessels, cells were expanded and maintained as a monolayer at 37 °C and subcultured weekly. The same culture media used for the monolayer cultures were used to grow the cells as 3D colonies.

Primary osteoblasts were maintained in complete medium (α-MEM (Cat. 41061037, GIBCO) supplemented with 10% FBS (Cat. A15.101, GE Healthcare) and 1% penicillin/streptomycin (Cat. P0781, Sigma-Aldrich). MC3T3-E1 osteoblastic cell line (ATCC) was cultured in complete medium (α-MEM (Cat. A1049001-01, GIBCO) supplemented with 10% FBS (Cat. A15.101, GE Healthcare) and 1% penicillin/streptomycin (Cat. P0781, Sigma-Aldrich).

Primary MSCs were isolated from lower hind limb muscle of 2- to 3-month old TIS7 WT and KO mice as described previously [43 (link)]. The generation of the TIS7 KO mouse, cell culture conditions, and reagents were described before [12 (link)]. Briefly, cells were proliferated in Ham’s F-10 medium without glutamine, supplemented with 20 % non-heat-inactivated fetal calf serum (FCS gold, GE Healthcare) and penicillin-streptomycin (GE Healthcare); 2.5 ng/mL recombinant bFGF-basic (PeproTech) was added freshly. Cells were differentiated in high-glucose DMEM, supplemented with 2 % non-heat-inactivated horse serum and penicillin-streptomycin (GE Healthcare). MSCs were grown on cell culture dishes coated with collagen from calf skin (Sigma).

K562 cells, kindly provided by Prof. Ewa Sitnicka Quinn, were maintained in HyClone RPMI-1640 Medium (GE Healthcare Life Sciences) supplemented with 10% heat-inactivated FBS (GE Healthcare Life Sciences) and 1% Penicillin–Streptomycin (GE Healthcare Life Sciences). CD34⁺ HSPCs were cultured in Serum-Free Expansion Medium (SFEM, STEMCELL Technologies) supplemented with 1% Penicillin–Streptomycin (GE Healthcare Life Sciences), stem cell factor (SCF), FLT3-ligand (FLT3L) and thrombopoietin (TPO). All cytokines were used in the concentration 100 ng/ml and acquired from Peprotech. CD34⁺ cells were thawed and pre-stimulated in culture medium at 37 °C, 5% CO₂ for 48 h prior to RNP electroporation and for 24 or 48 h prior to lentiviral sgRNA transduction.

Before the experiment, the cells were cultivated in DMEM Low-Glucose (5.5 mM) culture medium (Glutamine 3.24 mM, with bicarbonate) (Supplementary Table S4) (Sigma Life Science, Sigma-Aldrich, Gillingham, UK) with 10% fetal bovine serum (FBS) (Biowest, origin South America, Riverside, MO, USA) and penicillin-streptomycin (HyClone, GE Healthcare Life Sciences, Wien, Austria). The MDA-MB-468 cells were seeded in six-well plates at a density of 173,000 cells per well. For each well, 4 mL of DMEM, Low-Glucose culture medium (Sigma Life Science, Sigma-Aldrich, Great Britain) with 10% FBS (Biowest, origin South America, Riverside, MO, USA) and penicillin-streptomycin (HyClone, GE Healthcare Life Sciences, Wien, Austria) were added. The cell culture was maintained in a CO₂ incubator (Binder, Tuttlingen, Germany) under 5% CO₂ conditions at 37 °C and 80% humidity. Each time interval was prepared in triplicate and in separate wells. The final cell density at 72 h was 440,000 cells per well. The culture medium was not exchanged during the experiment. The medium was collected in 4- and 8-h time intervals.