Table S1. Hypoxia increases signaling through JNK in colon cancer cell lines in a cell-specific manner

Shown are the ratios of c-Jun phosphorylation during hypoxia to that of oxic control in each cell line. Data are based on the calculation of bands’ densities, normalized to actin, following Western blot of samples isolated in at least two independent experiments. Mean values are presented, pick values are highlighted.

Hypoxia (hours)Cell lines 1 3 5 24

BE 1.4 2.64 1.16 2.08Colo205 1.16 4.69 4.07 2.35DLD1 0.68 0.98 1.14 1.39HT29 1.72 2.28 2.37 0.9HCT15 0.71 0.64 0.72 1.02HCT116 1.74 0.69 1.09 0.4HCT116p53- 0.94 0.95 1.39 1.15KM12 1.62 1.88 3.54 1.46LoVo 1.17 1.73 2.83 0.6RKO 0.93 1.08 1.67 1.71SW480 2.02 1.56 1.21 2.59SW620 2.23 2.3 2.35 0.52

Table S2. Hypoxia increases resistance to chemotherapy in colon cancer cell lines

Shown are IC50 for oxaliplatin (mM), SN38 (nM) and 5-FU (mM) in a panel of colon cancer cell lines subjected to normal or hypoxic conditions for 24 hrs, followed by MTT assays as described in Materials and Methods. Table reflects data obtained in at least 3 independent experiments in triplicates.

Data were analyzed using Student’s t-test, P value less than 0.05 was accepted as a statistically significant difference when compared to corresponding oxic control. *, P < 0.05; **, P < 0.01, ***, P<0.001.

Oxaliplatin SN-38 5-FUOxic Hypoxic Oxic Hypoxic Oxic Hypoxic

BE 3.3 11*** 14 28*** 2.3 3.2*Colo205 0.9 3** 4.3 100*** 1.6 7.5*DLD1 6.7 13.5** 19 59** 9.3 21.9*HT29 0.62 1.67*** 15 27** 4.4 4.6HCT15 2.8 4* 40 100 4.8 4.9HCT116 0.9 0.95 2.5 5.5* 8.3 6.6HCT116p53- 5.2 5.6 8 25** 4.5 5.5*KM12 8 35*** 75 110** 8.8 9.9LoVo 1 1.2* 7 15** 1.3 4*RKO 1.05 1.3 4.7 11** 5.2 4.8SW480 0.7 2*** 22 60** 17 31.7**SW620 0.38 1.4*** 3.8 44*** 7.2 8

Figure S1. Effects of oxaliplatin and SN-38 on JNK pathway in oxic conditions. Assessment of the JNK signaling by oxaliplatin and SN-38 in colon cancer cell lines shows that it is not induced or altered in HT29 and SW620 cells, respectively, but activated in HCT116. Cells were treated with 5xIC50 of the corresponding drug and collected for Western blot analysis at several time points.

Table S3. Combination of the JNK inhibitor SP600125 with chemotherapy is synergistic in colon cancer cell lines

Cytotoxic interactions of JNK inhibitor SP600125 with oxaliplatin, SN-38 and 5-FU in colon cancer cell lines were evaluated in MTT-based assays. Combination indices (CI50) were calculated based on Chou-Talalay method using CompuSyn software. Presented data is an average value from 3 independent experiments in triplicates.

a,b P values are not significant when compared to the control (oxic) group; c P<0.01, significant when compared with control group.

Oxaliplatin SN-38 5-FU

Cell line CI oxic CI hypaCI oxic CI hypb

CI oxic CI hypc

HT29 0.97 0.89 0.78 0.79 0.91 0.58SW620 0.56 0.54 0.34 0.37 0.93 0.64HCT116 1 0.74 0.67 1.07 0.79 0.68LoVo 0.48 0.4 0.7 0.43 0.74 0.69KM12 0.5 0.57 0.63 0.62 0.97 0.85HCT15 0.76 0.86 0.69 0.72 0.85 0.43