Figure 1

1. Figure 1 HCT116-s HCT116-SN6 -S -A2 -SN50 -C8 -G7 NT SN38 NT SN38 [SN38] 24H SN38 1µM NT 0.1µM 1µM 5µM 10µM NT 1h 4h 7h 16h 24h 48h A pp38 pp38 HCT116-s HCT116-s tubulin tubulin pp38 pp38 HCT116-SN6 HCT116-SN6 tubulin tubulin B HCT116-SN6 C

2. HCT116-s HCT116-s p38α p38 b IC50 (nM) 3 p38 g p38δ 2 b-Actin 1 Sh p38 Sh Luc EV p38 CA 0 HCT116-SN6 Luc a b g d p38 CA isoforms p38 Sh RNA p38 Sh RNA p38α p38 b p38 g HCT116-SN6 HCT116-SN6 HCT116-SN6 25 25 16 p38δ 20 20 12 b-Actin 15 15 IC50 (nM) IC50 (nM) Sh p38 IC50 (nM) Sh Luc EV p38 CA 8 10 10 4 5 5 0 0 0 EV a + b EV a b g d Luc a b g d p38 CA isoforms  p< 0.05,  p< 0.01, p<0.001 Figure 2 A B C D HCT116-s HCT116-s 3 3 2 2 IC50 (nM) IC50 (nM) 1 1 0 0 EV a + b EV a b g d p38 CA isoforms p38 CA isoforms E F G H

3. 10 25 20 2,0 HCT116-SN6 HCT116-s NT 5-FU Oxali  8 20 15 1,5 pp38  6 15  SN38 IC50 (nM) SN38 IC50 (nm) SB IC50 (µM) SB IC50 (µm) 10 1,0  p38 4 10 b-Actin 0,5 5 2 5 0 0,0 0 0 SN38 SB SN38 +SB SN38 SB SN38 +SB SW480 HT29 7 12 14 SW480 HCT116-s HT29 HCT116-SN6 pATF2 6 12 10 4 HCT116-s 5 10 8 4 8 IC50 (nM) IC50 (nM) IC50 (nM) 3 6 3 6 NT SN38 SN38 + SB NT SN38 SN38 + SB 4 IC50 (µM) 2 2 4 HCT116-s 600 1 2 2 1 0 0 0 400 EV p38 a+b p38 a+b EV SN38 SN38+SB 0 IC50 (nM) p38 CA isoforms 5-FU 5-FU+SB p38 CA isoforms 12 200 10 0 8 Oxali Oxali+SB 6 4 2 0 SN38 SN38+SB  p< 0.05,  p< 0.01, p<0.001 Figure 3 A B D C IC50 (nM) F E

4. SB202190 Irinotecan 40 mg/ml IP Irinotecan 40 mg/ml IP 0 25 50 75 100 0 5 10 15 20 25 30 Days  p< 0.05,  p< 0.01, p<0.001 Figure 4 A B C % mice with tumor size doubled

5. 1 4 2 5 3 6 Figure 5 A B C

6. SW48-SN1 SW48-SN2 SW48-SN3 SW48-SN4 SW48-s pp38 tubulin A B Supplemental Figure 1: p38 is activated by phosphorylation in SN38-resistant SW48 cells. A: Drug sensitivity of the SW48 clones: IC50 values were determined using the SRB assay; the resistance factor was determined by dividing the IC50 value of each resistant clone by that of the sensitive clone SW48-s. Data represent the mean ±SD of at least 3 independent experiments. B: Western blot analysis of p38 phosphorylation in SW48-s cells and in the SN38 resistant clones SW48-SN1, SW48-SN2, SW48-SN3, SW48-SN4.

7. HCT116-s MKK6 CA EV 4 WB FLAG HCT116 3 2 IC50 (nM) pp38 WB 1 WB p38 0 EV MKK6 CA WB tubulin 8 SW480 SW480 7 6 EV MKK6 CA 5 Clone 1 Clone 2 4 IC50 (nM) 3 WB FLAG 2 1 0 EV MKK6 CA Clone 1 MKK6 CA Clone 2 pATF2 KA A B C D Supplemental Figure 2: Analysis of the involvement of MKK6 CA in SN38 resistance. A: Western blot analysis of HCT116 cells that express constitutively active (CA) MKK6 (or EV, as a control). MKK6 overexpression was detected with anti-FLAG antibody. p38 expression and activation were detected with anti-p38 and anti-phospho-p38 antibodies respectively. Equal loading is shown by tubulin expression. B: SRB assay on HCT116 expressing MKK6 CA or EV as control, and treated with SN38. C: Western blot analysis of 2 clones of SW480 cells that stably express constitutively active (CA) MKK6 (or EV, as a control). MKK6 overexpression was detected with anti-FLAG antibody. p38 activation was detected using a kinase assay to test p38a activity in the 2 clones stably expressing MKK6 CA or Empty Vector as a control. D: SRB assay on SW480 expressing MKK6 CA or EV as control, and treated with SN38.

8. shLuc Shp38α pMSCV pMSCVp38β shLuc Shp38α pMSCV pMSCVp38β HCT116-s HCT116-SN6 HCT116-SN6 SN38 treated samples pMSCV p38 b 140 NT 120 100 80 ICE arbitrary units 60 Topo I 40 Sn38 1µM 20 0 pMSCV p38 b 1.3 1.2 1.6 2.3 1.3 1.5 1.7 2.5 HCT116-SN6 SN38 treated samples 140 120 100 80 ICE arbitrary units HCT116-s HCT116-SN6 60 Sh Luc Shp38 a Sh Luc Shp38 a 40 20 0 NT ShLuc Shp38a ShLuc Shp38a HCT116-s HCT116-SN6 Sn38 1µM C A b-actin B Supplemental Figure 3: Analysis of Topoisomerase I expression and activity. A: Western blot analysis of Topoisomerase I (TopoI) in HCT116-s-ShLuc, HCT116-s-Shp38a, HCT116-s-pMSCV and HCT116-s-CAp38b cells and in HCT116-SN6-ShLuc, HCT116-SN6-Shp38a, HCT116-SN6-pMSCV and HCT116-SN6-CAp38b cells. Equal loading is shown by b-Actin expression. Numbers underneath the b-actin panel are the quantification data for total TopoI level obtained from western blot analysis after normalization to β-actin. B: Quantification ofSN38-induced TopoI-DNA complexes using the ICE bioassay and nuclear extracts from HCT-116-s-ShLuc and -Shp38a cells and HCT116-SN6-ShLuc and -Shp38a cells. The relative intensity of the immune complexes in SN38-treated cells was normalized to that of untreated cells. C: Quantification ofSN38-induced TopoI-DNA complexes using the ICE assay and nuclear extracts from HCT116-SN6-pMSCV and -CAp38b cells. The relative intensity of the immune complexes in SN38-treated cells was normalized to that of untreated cells.

9. 600 600 500 500 400 Tumor size (mm3) Tumor size (mm3) Tumor size (mm3) 300 400 1 2 3 4 5 6 7 8 Sh p38 a Sh Luc 200 HCT116-SN6 pMSCV 300 HCT116-SN6 p38 b 100 HCT116-SN6 pMSCV 200 HCT116-SN6 p38 b CA 0 Days 0 5 10 15 20 25 HCT116-SN6 Sh Luc Days Days 100 HCT116-SN6 Sh p38 a Irinotecan 40 mg/ml IP Irinotecan 40 mg/ml IP 0 0 5 10 15 25 E 20 p38 b b-actin p38 a 600 1 2 3 4 5 6 7 8 p38 beta b-actin pMSCV 500 400 HCT116-SN6 Sh Luc p38 a /b-actin 300 HCT116-SN6 Sh p38 a 60 Arbitrary units 50 200 40 30 100 20 10 0 0 0 5 10 15 20 25 Sh p38 alpha Sh Luc HCT116-SN6 A B C D Supplemental Figure 4: Thedifferential expression of the four p38 isoforms influences the response to irinotecan. A: Tumor growth kinetics in mice xenografted with HCT116-SN6- pMSCV or HCT116-SN6-p38b, HCT116-SN6-ShLuc or HCT116-SN6-Shp38a cells before irinotecan treatment. B: Tumor growth kinetics in mice xenografted with HCT116-SN6-ShLuc or HCT116-SN6-Shp38a cells and treated with irinotecan. C: Tumor growth kinetics in mice xenografted with HCT116-SN6-pMSCV or HCT116-SN6-p38b cells and treated with irinotecan. D: Western blot analysis of p38b expression in HCT116-SN6-pMSCV and HCT116-SN6-CA38b xenografts. Equal loading is shown using b-Actin. E: Western blot analysis of p38a expression in HCT116-SN6-ShLuc or HCT116-SN6-Shp38a xenografts. Equal loading is shown using b-Actin. Histogram shows the quantification data for p38a level obtained from western blot analysis after normalization to β-actin.