Calcusyn v2

Cells (5,000/well) were added to 96‐well plates. The EC50 concentration of luteolin in normal liver cell LO2 for 48 h was 11.96 µg/ml, which was detected by us. As a result, 5 μg/ml luteolin was considered as a safe dose and used in the following experiments. Then, the normal liver cells and liver cancer cells were infected using VV‐IL‐24 (4 MOI) and luteolin (5 µg/ml) or both VV‐IL‐24 (4 MOI) and luteolin (5 µg/ml). Cells were incubated for 24, 48, 72 or 96 h after which 20 µl MTT (Macklin) was added per well for 4 h at 37°C. Supernatants were then removed, and 150 µl DMSO (Amresco) was added per well. Plates were agitated for 10 min, after which absorbance at 490 nm was assessed via Microplate Reader (Tecan Group, Ltd., Mannedorf, Switzerland). CalcuSyn v2.1 (Biosoft, Cambridge, UK) was used to calculate combination index (CI) values, and average CIs from three independent experiments were designed as X‐mark on the graph. when CI = 1 indicates an additive relationship, CI > 1 indicates antagonism, and CI < 1 indicates synergy.²⁰

To quantify the synergistic activity of APR-246 with system x_C⁻ inhibitors, the GI50 dose of single agents was firstly determined by fitting the Hill equation using Prism 6 software (Graphpad). Cells were then treated for 96 h with combinations of APR-246 and system x_C⁻ inhibitors over a range of concentrations held at a fixed ratio and based on the GI50 of each drug. The highest and lowest combination ratios were three times and 1/10th the GI50, respectively. Combination indexes (CI) at 50%, 75% and 90% reduction in cell viability was determined using CalcuSyn v2 (Biosoft) where synergism: CI<0.9, antagonism: CI>1.1 and additive effect: 0.9≤CI≤1.1 (ref. 43 (link)).

For in vitro drug combination studies, IC50 values and combination indices were determined by CalcuSyn v2 (BioSoft), as previously described [106 (link)]. A Bliss independence model was used to evaluate combination effects on an efficacy scale [108 (link)]. The Bliss expected value was calculated using the equation (A + B) – (A × B), in which A and B are the percentage of growth inhibition induced by agents A and B for a given pair of doses. Values of the difference between the Bliss expected growth inhibition and the observed growth inhibition of approximately 0% indicate additivity, >0% indicate synergy, and <0% indicate antagonism. Results are reported as mean ± SD. Data were considered significant at p < 0.05. Saos2 flank tumor xenograft and TT2-77 PDX tumor volume data were analyzed by a two-way repeated measures ANOVA (one factor repetition), followed by a Holm–Sidak post-hoc pairwise multiple comparisons test using GraphPad Prism Software (GraphPad, Inc., San Diego, CA, USA) and SigmaPlot 11.2 (Systat Software, Inc., San Jose, CA, USA). The tumor volume is presented as the mean ± SEM and was determined 3× per week. Saos2 CDX and TT2-77 PDX survival was assessed by Kaplan–Meier plots and analyzed by the log-rank test.

Seventy-two hour growth inhibition assays and GI₅₀ concentration determination were performed as previously described [38 (link)]. For combination studies, cells were treated for 72 hours with RG7388 and conventional chemotherapies alone and in combination simultaneously at constant 1:1 ratios of 0.25×, 0.5×, 1×, 2× and 4×, or 0.125×, 0.25×, 0.5×, 1× and 2×, their respective GI₅₀ concentrations, depending on the drug solubility. Median-effect analysis and Combination Index (CI) values were determined using CalcuSyn v2 (Biosoft, Cambridge, UK). Experiments were at least n=3.

MTT assay was performed as described previously (18 (link)). Colony formation was performed as described elsewhere (19 (link)). IC50 values were obtained using the GraphPad Prism software. CalcuSyn drug dose-effect analysis software (CalcuSyn V2, Biosoft, Cambridge, UK) was used to determine synergy (combination indexes and isobolograms).

Cells were seeded in 96-well plates (Corning, VWR International Ltd., Lutterworth, UK), and allowed to adhere overnight before treatment with ATR or PARP inhibitors alone or in combination for 72 h. Inhibitors were added at 200× dilution to give a final DMSO concentration of 0.05%. Percentage control growth was assessed using the XTT cell proliferation assay (Roche, Burgess Hill, UK) according to the manufacturer’s instructions and using the following formula: (average absorbance test/average absorbance control) × 100. Combination Index (CI) values were determined by the Chou–Talalay method using CalcuSyn v2 (Biosoft, Cambridge, UK).