T-REx Flp-In DLD-1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher) containing 10% tetracycline-free fetal bovine serum (FBS) (Omega Scientific) and 100 U/mL penicillin-streptomycin. hTERT-immortalized RPE-1 cells were cultured in DMEM/F12 (Thermo Fisher) supplemented with 10% FBS, 100 U/mL penicillin-streptomycin, 2 mM L-glutamine, and 0.348% sodium bicarbonate. All cells were maintained at 37°C under 5% CO2 and atmospheric oxygen.
Doxycycline (DOX) and indole-3-acetic acid (IAA) were purchased from Sigma, dissolved in cell culture-grade water, and used at 1 μg/mL and 500 μM, respectively. Where indicated, DOX/IAA washout was performed by two washes in PBS followed by addition of growth medium. The Mps1 inhibitor reversine (Santa Cruz) was dissolved in DMSO and used at the indicated concentrations. Geneticin (G418 Sulfate) and hygromycin (Thermo Fisher Scientific) were used at selection concentrations of 300 μg/mL and 200 μg/mL, respectively. DLD-1 siRNA transfections were conducted with Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) using characterized siRNAs (GE Healthcare Dharmacon) as previously described33 (link).
Doxycycline (DOX) and indole-3-acetic acid (IAA) were purchased from Sigma, dissolved in cell culture-grade water, and used at 1 μg/mL and 500 μM, respectively. Where indicated, DOX/IAA washout was performed by two washes in PBS followed by addition of growth medium. The Mps1 inhibitor reversine (Santa Cruz) was dissolved in DMSO and used at the indicated concentrations. Geneticin (G418 Sulfate) and hygromycin (Thermo Fisher Scientific) were used at selection concentrations of 300 μg/mL and 200 μg/mL, respectively. DLD-1 siRNA transfections were conducted with Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) using characterized siRNAs (GE Healthcare Dharmacon) as previously described33 (link).