Glutamax

Monocyte Medium: RPMI 1640 (Gibco), 10% FCS (Gibco), 10 mM Hepes (Gibco), 2 mM Glutamax (Gibco), 1% Antibiotic/Antimycotic (Gibco); StemPro Differentiation Medium (SP): KnockOut DMEM/F-12 (Gibco), 2% StemPro Neural Supplement (Gibco), 2 mM Glutamax (Gibco), 1% Antibiotic/Antimycotic (Gibco); OPC Differentiation Medium (OPC Diff): OPC Spontaneous Differentiation Media Kit (Sigma-Aldrich SCM106); OPC Expansion Medium: Human OPC Expansion Culture Media Kit (Sigma-Aldrich SCM107);
Neural Differentiation Medium (NDM): Neurobasal Medium (Gibco), 2% B27 Supplement (Gibco), 2 mM Glutamax (Gibco), 1% Antibiotic/Antimycotic (Gibco); B27 Medium: KnockOut DMEM/F-12 (Gibco), 2% B27 Supplement (Gibco), 2 mM Glutamax (Gibco), 1% Antibiotic/Antimycotic (Gibco); N2 Medium: KnockOut DMEM/F-12 (Gibco), 1% N2 Supplement (Gibco), 2 mM Glutamax (Gibco), 1% Antibiotic/Antimycotic (Gibco); KO DMEM/F-12 Medium: KnockOut DMEM/F-12 (Gibco), 2 mM Glutamax (Gibco); 1% Antibiotic/Antimycotic (Gibco); RPMI 1640 Medium: RPMI 1640 (Gibco), 2 nM Glutamax (Gibco), 1% Antibiotic/Antimycotic (Gibco).

The colon carcinoma HCT116 cell line was cultured in McCoy’s 5A (modified) medium supplemented with GlutaMAX™ (ThermoFisher), 10% fetal bovine serum (Gibco), and penicillin-streptomycin (Gibco). Breast cancer cell line MCF7 used for the gene expression analysis was cultured in Minimum Essential Eagle medium with 1 mM sodium pyruvate medium supplemented with GlutaMAX™ (ThermoFisher), 10% fetal bovine serum, and penicillin-streptomycin. HEK293 Rpn11-HTBH cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with GlutaMAX™ (ThermoFisher), 10% fetal bovine serum, and penicillin-streptomycin. B16-F10 mouse melanoma cells (ATCC) were maintained in DMEM (Lonza) supplemented with 10% FBS (Gibco) and 2 mM L-glutamine (Lonza). The cells were maintained at 37°C in a 5% CO₂ humidified incubator and were regularly passaged at 70-80% confluence. Treatments with b-AP15(Vivolux AB), bortezomib (Sigma), and chloroquine (Sigma) were all carried out at 70-80% confluence. All the compounds used were dissolved in DMSO. Treatment and control groups were exposed to an equivalent amount of DMSO (0.1-0.5%).

Hepatic differentiation from hiPSC (RIKEN2F) was performed as described previously, with some modifications. Human iPSCs were passaged and then seeded at a cell density of 25,000 cells/cm² on iMatrix511-coated 6-well plate in Stemfit^® and cultured for 2 days. Subsequently, to initiate differentiation, the cells were treated with RPMI-1640 plus 2% B27 Minus Insulin (RPMI-B27) containing 100 ng/ml Activin A (Nacali Tesque), 6 µM CHIR-99021 (MCE), and 1% GlutaMAX^® (Thermo Fisher Scientific) for 24 h, followed by RPMI-B27 with 50 ng/ml Activin A for 24 h. For differentiation into hepatic progenitors, the cells were treated with RPMI-B27 containing 1% GlutaMAX^® and 10 ng/ml BMP-4 (Thermo Fisher Scientific) for 4 days. The medium was changed to hepatocyte maturation medium: Leibovitz’s L-15 medium (Fuji Film Wako Chemical Inc.) containing 8.3% tryptose phosphate broth, 10 μM hydrocortisone 21-hemisuccinate, 50 μg/ml sodium L-ascorbate, 100 nM dexamethasone (DEX) (all from Merck), 0.58% insulin-transferrin-selenium (ITS), 2 mM GlutaMAX^® (all from Thermo Fisher Scientific), 8.3% fetal bovine serum (Biowest, FL, United States), and 100 nM Dihexa (TRC, Ontario, Canada). This culture was continued for 17 days to obtain definitively differentiated hepatocyte-like cells. During the culture, media change was performed every 2 days.

The HEK-293 (ATCC, CRL-1573) and HeLa (ATCC, CCL-2) cells were cultured in DMEM (4.5 g/L glucose, Thermo Fisher Scientific) supplemented with 2 mM GlutaMAX (Thermo Fisher Scientific), 10% FCS (Thermo Fisher Scientific), 100 U/mL of penicillin, and 100 µg/mL of streptomycin (Thermo Fisher Scientific). The PC12 Tet-Off cells (Clontech, 631134; PC12-TO) were maintained in DMEM medium (1 g/l glucose, Thermo Fisher Scientific) supplemented with 10% FCS, 5% horse serum (HS), 2 mM GlutaMAX, 100 U/mL penicillin, and 100 µg/mL streptomycin (all Thermo Fisher Scientific). The osteosarcoma U-2 OS cells (ATCC, HTB-96) were cultured in McCoy’s 5A (Modified) Medium supplemented with GlutaMAX containing 10% FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin (all Thermo Fisher Scientific). The PC12-TO cells were grown on surfaces coated with poly-l-lysine (PLL, Sigma-Aldrich) for the maintenance and experiments. For coating, the plates were incubated with PLL (0.02 mg/mL final concentration diluted in ddH₂O) for 30 min at 37 °C, washed twice with ddH₂O, and air-dried. The cells were cultured at 37 °C and 5% CO₂.

Cultures of A549 lung alveolar epithelial cells (from Max Gutierrez) and human foreskin fibroblast cells (ATCC #SCRC-1041) for maintenance of Toxoplasma were grown in Gibco DMEM with GlutaMAX (Thermo Fisher Scientific #12077549) supplemented with 10% heat-inactivated FBS (Life Technologies) and cultured at 37°C in 5% CO₂. Human umbilical vein endothelial cells (Promocell #C-12203) were cultured in Gibco Medium 199 (Thermo Fisher Scientific #11554426) supplemented with 20% heat-inactivated FBS, Heparin (Merck), and endothelial cell growth supplement (Merck #02-102). THP-1 monocyte cell line (TIB202, ATCC) was maintained in Gibco RPMI with GlutaMAX (Thermo Fisher Scientific #61870036) with 10% heat-inactivated FBS. THP1s were differentiated to macrophages with 50 ng/mL phorbol myristate acetate (PMA) (Merck #P1585) for 3 days and then rested for 2 days in PMA-free, complete medium.
Toxoplasma gondii stably expressing luciferase/EGFP or Td-tomato (Prugniaud type II and CEP type III) were maintained in vitro by serial passage on monolayers of HFF cells, cultured in Gibco DMEM with GlutaMAX (Thermo Fisher Scientific) supplemented with 1% FBS at 37°C in 5% CO₂. All cell culture was performed without the addition of antibiotics. Cell lines and parasites were routinely tested for mycoplasma contamination by PCR test.

All cells were cultured and maintained at 37 °C with 5% CO₂. Antibiotics were not used for cell culture. HEK293T cells (ATCC CRL-3216) and HeLa cells (ATCC CCL-2) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) plus GlutaMax (ThermoFisher) supplemented with 10% (v/v) fetal bovine serum (FBS). K562 cells (ATCC CCL-243) were cultured in Roswell Park Memorial Institute (RPMI) 1640 Medium plus GlutaMax (ThermoFisher) supplemented with 10% (v/v) FBS. U2OS cells were cultured in MyCoy’s 5A Medium plus GlutaMax (ThermoFisher) supplemented with 10 % (v/v) FBS.
hiPS cells (human episomal iPS cell line; A18945; ThermoFisher) were cultured in Essential 8 Flex Medium (ThermoFisher) supplemented with RevitaCell after passaging (ThermoFisher) according to the manufacturer’s directions. Versene (Thermo Fisher) was used for cell passaging and dissociation. Prior to nucleofection, cells were harvested with Accutase (ThermoFisher).
For data shown in Fig. 5i, j, nuclease expression plasmids were constructed whereby the Cas-enzyme construct (Cas9 or RDN(A89E)) was proceeded by P2A-GFP to enable isolation of transfected cells. iPS cells were flow sorted at the MIT FACS core 3–5 days after nucleofection and genomic DNA was isolated directly after sorting.