Cell viability assays were conducted in the HCT116 parental cell line and the method of Chou and Talalay was used to evaluate the interaction between (A) GalR1 siRNA (siGALR1) or (B) galanin siRNA (siGAL) and increasing concentrations of either 5-FU or oxaliplatin at 48/72h. CI values <1, =1, and >1 indicating synergism, additivity, and antagonism, respectively. GALR1 silencing was confirmed by Western blot (A, inset) and galanin silencing was confirmed by Q-PCR (B, inset). (C) Cell viability of HCT116 oxaliplatin-resistant (OXR) and 5-FU-resistant (FUR) cell lines following galanin/GalR1 silencing (10nM siRNA) for 24h prior to 48h co-treatment with parental IC30(48h) and IC50(48h) doses of chemotherapy. A synergistic interaction was determined by 2-way ANOVA. *P<0.05; ***P<0.001 (D) Annexin V/PI flow cytometric analysis was used to measure apoptosis levels in HCT116 parental cells following galanin/GalR1 silencing (5nM siRNA) for 24h prior to a 24h co-treatment with IC30(48h) doses of either 5-FU or oxaliplatin. (D, inset) Western blot showing PARP cleavage in HCT116 cells transfected with 5nM siGALR1 for 24h prior to a 24h co-treatment with ~IC30(48h) doses of either 5-FU or oxaliplatin. Results are given as mean values ± SEM of triplicate measurements. *P<0.05; **P<0.01; ***P<0.001. SC=siRNA control.