On targetplus human myc smartpool

The LNCaP-MYC (Ramos-Montoya et al., 2014 (link)) and the corresponding empty vector (EV) line were cultured at 37 °C and 5% CO₂ in RPMI1640 (Gibco, 21875), containing 10% fetal bovine serum (FBS) (Gibco, 10500) and 2 μg/ml puromycin and 200 μg/ml G418 (Gibco, 10131019) for plasmid maintenance. For hormone starvation, cells were washed once with PBS (Gibco, 10010) and cultured in phenol red-free RMPI1640 (Gibco, 11835063), containing 10% charcoal-stripped FBS (Gibco, 12676029) for 72 h before starting the experiment. MYC overexpression was induced using 2 μg/ml doxycycline. Parental LNCaP cells were cultured under the same conditions minus the antibiotics. VCaP cells were cultured in DMEM (Gibco), containing 10% FBS under the same conditions.
For viability assays, the amount of viable cells was determined using Cell Aequous solution MTS reagent (Promega, G3581) following the manufacturer's recommendations.
Sarcosine levels were determined using a Sarcosine Assay Kit (Abcam, ab65338) following the manufacturer's recommendations.
Reverse siRNA transfection was performed using the Lipofectamine RNAiMAX transfection reagent (Life technologies, 13778150) and OptiMEM transfection medium (Life technologies, 31985-070). The following siRNAs were used: ON-TARGETplus Non-Targeting Pool (Thermo Scientific, D-001810-10) and ON-TARGETplus Human MYC SMARTpool (Thermo Scientific, L-003282-02).