Cell viability and RNA interference (siRNA knockdown) assays

To quantify cell viability, the CellTiter-Glo Cell luminescence-based viability assay (Promega, Madison, WI) was used according to manufacturer’s protocol and as previously described (29). Luminescence was measured using a Fluoroskan Ascent FL luminometer (Thermo Scientific, Rockford, IL) to obtain triplicate sample 24, 48 and 72 h time point determinations after treatment, normalized to that of control cells and graphically displayed as mean (+/− SD) percent control values. Coefficient of drug interaction (CDI) values were determined as previously described by us and others (29, 30), with CDI = %viability(drugA+drugB)/%viability(drugA) × %viability(drugB).

The ON-TARGET plus SMART pool of four different siRNA oligonucleotides to PRODH (J-009543–05-0002, J-009543–06-0002, J-009543–07-0002, and J-009543–08-0002) and a non-specific control siRNA (D-001810–01-05) were all obtained from Dharmacon (GE Dharmacon, Lafayette, CO). Pooled and single oligos were transfected into replicate cell cultures using Lipofectamine 2000 (ThermoFisher Scientific) as we have previously reported (31, 32).