Maintenance and Genetic Manipulation of Cell Lines

HeLa cells were ordered from ATCC and maintained in RPMI 1640 medium, and HAP1 cells were purchased from Horizon Discovery and maintained in Iscove’s Modified Dulbecco’s medium. The ID8 mouse ovarian cancer cells were kindly provided by Dr. Guang Peng’s laboratory (MD Anderson Cancer Center). HeLa GRB2 deficient cells and HAP1 GRB2 deficient cells were generated by using CRISPR/Cas9 system as described previously^{11 (link)}. ID8 GRB2 stably knocked-down cells were generated by using GRB2 shRNAs which were purchased from Dharmacon (RMM4431-200333332, RMM4431-200335970, RMM4431-200399380, RMM4431-200404712). Media were supplemented with 10% fetal bovine serum (Lonza) and 1% antibiotic/antimycotic (Lonza) except The ID8 cells were maintained in DMEM supplemented with 4% fetal bovine serum (Lonza), 5 μg/mL insulin, 1% penicillin/streptomycin, 5 μg/mL transferrin, and 5 ng/mL sodium selenite. Cells were incubated at 37 °C in a humidified incubator with 5% CO2. Mycoplasma testing of these cell lines has confirmed negative results.