Additional file 2 of Complex interactions of lovastatin with 10 chemotherapeutic drugs: a rigorous evaluation of synergism and antagonism

Additional file 2: Legends Figure S1. Schematic diagram of the 96-well plate for the dual-drug assays (see Methods). The dose-responses (Columns 9 and 10), which are required for the computation of Loewe additive synergy, are performed at the same starting concentrations and with the same dilutions as in the dual-dilution portion of the plate. The format shown is compatible with the Combenefit software. BL = Plate blank (synthetic complete media + dextrose only). Figure S2. Tamoxifen plus lovastatin data on day 3. The comparable data for day two are depicted and described in Figure 2. The synergy matrix shown here is very similar to the day two matrix. Figure S3. Doxorubicin and lovastatin interactions on day three. The data are quite similar to the results on day two, shown in Figure 3. Figure S4. Methotrexate and lovastatin on day three. The patterns are quite similar to those recorded on day two (Fig. 4). The Loewe synergy matrix (D) is the same plot as shown in Fig. 4e. Figure S5. Experimental dose-response data for the combination of rapamycin and lovastatin. The combination dose-response data are visualized as contours, with 25, 50, 75 and 100% of control levels. Even though the EC50 of rapamycin is three-fold greater on day3 (Fig. 5, d vs. a), the 25% dose-response contour is achievable at all concentrations of rapamycin with only a modest increase in lovastatin concentration compared to day two. These data, supportive of the increase in synergy seen in the Loewe matrix (Fig. 5f compared to c), suggest that lovastatin somehow prolongs the efficacy of rapamycin. Figure S6. Day three Loewe synergy matrices corresponding to the day two data shown in Fig. 6. Epothilone plus lovastatin exhibits significant synergy on day three (C) whereas the combination did not on day two (Fig. 6c). The matrix for 5-fluoruracil (A) shows no significant interactions, as it did on day two. The other four drugs, gemcitabine (B), cisplatin (D), cyclophosphamide (E), and etoposide (F), exhibit greater antagonism on the third day. Figure S7. Loewe synergy matrices on days two and three for three cell lines in dual-drug assays of cisplatin in combination with lovastatin. Two human cancer cells lines, A549 (lung adenocarcinoma) (A, D) and MCF7 (breast cancer) (C, F), exhibited significant antagonism (and no significant synergism) on day two; the antagonism was more pronounced on day three. The HT29 colon adenocarcinoma cell line exhibited synergism and antagonism, both statistically significant, on day two (B) and to a lesser degree, on day three (E). The data shown here are comparable to that obtained with the S. cerevisiae assays (see Table 2, and Figs. 6c and S6D).

附加文件2：图S1图例。双药物测定用96孔板示意图（详见方法部分）。用于计算洛维（Loewe）相加协同作用所需的剂量反应曲线（第9、10列），采用与板上双稀释区域一致的初始浓度与稀释梯度进行实验。本图示格式可与Combenefit软件兼容。BL代表板空白对照（仅含合成完全培养基与葡萄糖）。
图S2：第3天他莫昔芬联合洛伐他汀的实验数据。第2天的对应数据已在图2中展示与说明，本研究的协同矩阵与第2天矩阵高度相似。
图S3：第3天阿霉素与洛伐他汀的药物相互作用数据，其结果与图3展示的第2天实验数据高度一致。
图S4：第3天甲氨蝶呤与洛伐他汀的实验结果，其变化模式与第2天记录的结果（图4）高度相似。其中洛维协同矩阵（D）与图4e展示的图像完全一致。
图S5：雷帕霉素联合洛伐他汀的实验剂量反应数据。联合用药的剂量反应数据以等高线图可视化，分别对应对照组水平的25%、50%、75%与100%。尽管第3天时雷帕霉素的半最大效应浓度（EC50）较第2天升高3倍（图5d与图5a对比），但与第2天相比，仅需适度提高洛伐他汀浓度，即可在所有雷帕霉素浓度下达到25%的剂量反应等高线阈值。本数据支持洛维协同矩阵中观察到的协同作用增强现象（图5f与图5c对比），提示洛伐他汀可在一定程度上延长雷帕霉素的药效持续时间。
图S6：对应图6展示的第2天数据的第3天洛维协同矩阵。第3天时，埃坡霉素联合洛伐他汀表现出显著协同作用（面板C），而该组合在第2天时未表现出协同作用（图6c）。5-氟尿嘧啶（A）的矩阵未显示出显著药物相互作用，与第2天结果一致。其余4种药物，即吉西他滨（B）、顺铂（D）、环磷酰胺（E）与依托泊苷（F），在第3天时均表现出更强的拮抗作用。
图S7：3种细胞系在顺铂联合洛伐他汀的双药物测定中，第2天与第3天的洛维协同矩阵。2种人类癌细胞系：A549（肺腺癌，面板A、D）与MCF7（乳腺癌，面板C、F）在第2天时表现出显著拮抗作用（无显著协同作用），且该拮抗作用在第3天时更为明显。HT29结肠腺癌细胞系在第2天（面板B）同时表现出具有统计学意义的协同与拮抗作用，而在第3天时（面板E）该现象程度有所减弱。本研究展示的数据与酿酒酵母（S. cerevisiae）测定获得的结果具有可比性（详见表2、图6c与图S6D）。