SUPPORTING INFORMATION

Inhibition of p38 MAPK sensitizes tumor cells to cisplatin-induced apoptosis mediated by reactive oxygen species and JNK

Lorena Pereira, Ana Igea, Begoña Canovas, Ignacio Dolado and Angel R. Nebreda

Table of Content Table S1 Table S2 Figure S1 Figure S2 Figure S3 Figure S4 Figure S5 Figure S6 Figure S7 Figure S8 Figure S9 Figure S10 Figure S11 Figure S12

Table S1. Antioxidant -encoding regulated by p38 MAPK in cancer cells

RefSeq Name Array MCF7 HT-29

NM_002083 GPX2- Glutathione 2 -1.91 - 1.38 1.83

NM_002084 GPX3- 3 -1.49 - 1.07 - 1.31

NM_001509 GPX5- Glutathione peroxidase 5 -1.96 -2.16 -1.50

NM_000637 GSR- Glutathione reductase -1.70 -1.17 1.09

NM_006151 LPO- -1.88 1.50 - 5.56

NM_000963 PTGS2/COX2 - Prostaglandin-endoperoxide synthase 2 -5.87 -4.09 -1.53

NM_144651 PXDNL- Peroxidasin homolog -1.86 -9.52 -1.45

NM_032243 TXNDC2- Thioredoxin domain containing 2 -3.15 -1.91 -2.35

The expression of eight candidate genes identified in the array was validated by RT-PCR in breast MCF7 and colon HT-29 cancer cells. Numbers indicate fold changes in the expression levels of the genes upon incubation of the cells with SB203580 to inhibit p38 MAPK signaling. Genes downregulated at least 1.5 fold in both cell lines are shadowed

Table S2. Sequences of primers used for RT-PCR analysis

Refseq Primers sequences

NM_002083 GPX2 FW: 5’- CCTCCCCACCCCTCTAATAG - 3’ RV: 5’- TCTACCTTCTCCCCATCCAG - 3’ NM_002084 GPX3 FW: 5’- TGTCAATGGAGAGAAAGAGCAG - 3’ RV: 5’- TCTCAAAGTTCCAGCGGATG - 3’ NM_001509 GPX5 FW: 5’- ACATCCTGGCGTACTTGAAG - 3’ RV: 5’- GGGAGAGCAAGTCATTAGGTG - 3’ NM_000637 GSR FW: 5’- CAGGGACTTGGGTGTGATG - 3’ RV: 5’- ACGTGTCTCCTGGTTCTCA - 3’ NM_006151 LPO FW: 5’- TGAAAAGACAAGGCACTGGG - 3’ RV: 5’- TGAAGCAAGATGAGGGAAGC - 3’ NM_000963 PTGS2 FW: 5’- ACAGGCTTCCATTGACCAG - 3’ RV: 5’- TCACCATAGAGTGCTTCCAAC - 3’ NM_144651 PXNDL FW: 5’- CCAAGTGATTCCCCAGAGAAG -3 ’ RV: 5’- GTTGCTTAAGTCAGTGGTTTCC - 3’ NM_032243 TXNDC2 FW: 5’- TGATGCCAGTGAATGCGTAG - 3’ RV: 5’- CGTTGCTGGACAGGACTAG - 3’ NM_002827 PTPN1 FW: 5’- AGAAGGACGAGGACCATGCAC - 3’ RV: 5’- AGTGGAGGAGGGTCAGGCTAT - 3’ NM_002835 PTPN12 FW: 5’- GATGGTGCTGTGACCAGGAAC - 3’ RV: 5’- TCATGTCCATTCTGAAGGTGG - 3’ NM_004420 DUSP8 FW: 5’- GATGACGCAAAATGGAATAAGC - 3’ RV: 5’- CTTCACGAACCTGTAGGCG - 3’ NM_144729 DUSP10 FW: 5’- TGAACATCGGCTACGTCATC - 3’ RV: 5’- TGGTGTAAGGATTCTCGGTG - 3’ NM_030640 DUSP16 FW: 5’- CAGAATGGGATTGGTTATGTG - 3’ RV: 5’- TAGGCGATAGCGATGGTGG - 3’

A C CDDP - + - + PH p85PARP - 1 5 10 SB (μM) P-JNK P-JNK

JNK JNK P-p38 Tubulin P-MK2 MCF7 tubulin SW620 B

PH CDDP CDDP + PH

2.2 2.8 21.3 31.5

HT29

4.6 5.0 5.0 7.2 PI

0.8 1.5 6.4 13.0

SW620

5.7 2.5 5.4 8.5

Annexin V

Figure S1. A. MCF7 cells were treated with increasing concentrations of SB203580 (SB, 1- 10 μM) for 6 h. Total cell lysates were analyzed by immunoblotting with the indicated antibodies. B. HT-29 and SW620 cells were pre-incubated for 2 h with PH-797804 (PH, 2 μM) and then treated with cisplatin (CDDP, 100 μM) for 24 h. Cell death was measured with propidium iodide (PI) and Annexin V staining. The percentages of apoptotic cells are indicated. C. SW620 cells were incubated overnight with PH-797804 (PH, 2 μM) and then treated for 8 h with cisplatin (CDDP, 100 μM). Total cell lysates were analyzed by immunoblotting with the indicated antibodies. A C

shControl shJNK1 shJNK2 α β α + sip38β α β α + sip38β - + + - + + - + + CDDP siControlsip38 sip38 sip38 siControlsip38 sip38 sip38 - - + - - + - - + SB - - - - + + + + CDDP p38β p85PARP p38α JNK P-JNK P-HSP27

JNK HSP27

GAPDH Tubulin MCF7 HT-29

B CDDP CDDP + PH

siControl 3.0 12.1 17.6

siControlsiJNK1/2 2.1 23.1 37.3 JNK PI

Tubulin

HT-29 3.5 8.9 12.4

siJNK1/2

2.2 19.9 21.3

Annexin V

Figure S2. A. MCF7 cells were transfected with siRNAs (50 nM) either control or against p38α, p38β or both together. After 48 h, cells were treated for 8 h with cisplatin (CDDP, 100 μM). Total lysates were analyzed by immunoblotting with the indicated antibodies. The upper blot was incubated first with the p38β antibody and then with the p38α antibody. B. HT-29 cells were transfected with siRNAs (50 nM) either control or against JNK1/2. After 48 h, cells were incubated for 2 h in the presence or absence of PH-797804 (PH) and then treated for 24 h with cisplatin (CDDP, 100 μM). Cell death was quantified by staining with propidium iodide (PI) and Annexin V. Total cell lysates were analyzed by immunoblotting with the indicated antibodies (right panel). C. HT-29 cells were infected with lentiviruses expressing shRNAs against JNK1 or JNK2 or a non-targeting control. Pools of cells were incubated overnight in the presence or absence of SB203580 (SB, 10 μM) and then treated for 8 h with cisplatin (CDDP, 100 μM). Total cell lysates were analyzed by immunoblotting with the indicated antibodies. A HT-29 DMSO SB 6h SB o/n

H2O2

MCF7

cell number DMSO SB 1h SB 6h

H2O2

DCFDA

B AOX C CDDP SB +SB H₂O₂ - - + AOX - + - + - + - + Derivatized - + + PH p85PARP

97 P-JNK 68 Oxyblot JNK 43

29 60% 80% 60% 100% (Oxy/Tub) P-HSP27 kDa Tubulin Tubulin MCF7 MCF7

Figure S3. A. HT-29 and MCF7 cells were incubated for the indicated times with SB203580 (SB, 10 μM), overnight with DMSO, or 10 min with H2O2 (5 mM). ROS levels were measured with DCFDA by flow cytometry. B. MCF7 cells were pre-treated for 1 h with a mixture of NAC and GSH antioxidants (AOX) and then incubated overnight with SB203580 (SB, 10 μM), or were treated with 5 mM H2O2 for 1 h, as indicated. Cell lysates were analyzed by immunoblotting using Oxyblot and the total protein oxidation signal per lane was quantified using tubulin as a reference. C. MCF7 cells were pre-treated for 1 h with antioxidants (AOX) as in (B), followed by overnight incubation with PH-797804 (PH, 2 μM) and then treated with cisplatin (CDDP, 100 μM) for 8 h. Total cell lysates were analyzed by immunoblotting with the indicated antibodies. A HT-29 SW620 MCF7 n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. n.s. 0.6 0.6 1.0 0.4 0.4 0.5 0.2 0.2 absorbance absorbance absorbance

0 0 0 - SB AOX - SB AOX - SB AOX

B MCF7 HT-29

siCtrl #1 #2 siCtrl #1 #2 p38α Tubulin

*** * n.s. *** n.s. * ** *** *** 0.5 0.20 *** *** *** 0.4 0.15 0.3

0.10 0.2 absorbance absorbance

0.05 0.1

siCtrl #1 #2 siCtrl #1 #2 MCF7 sip38α HT-29 sip38α

Figure S4. A. Cells were treated for 1 h with a mixture of NAC and GSH antioxidants (AOX) or incubated overnight with SB203580 (SB, 10 μM) and then plated for clonogenic assays. Colonies were dissolved in methanol and the absorbance reads (540 nm) are represented in the bar diagrams. B. Cells were incubated for 48 h either with two different siRNAs against p38α (#1 and #2) or with a scrambled siRNA (siCtrl) as a control. Total cell lysates were analyzed by immunoblotting with the indicated antibodies (upper blot). For clonogenic assays, siRNA-treated cells were treated for 1 h with AOX as in (A) followed by 1 h incubation with cisplatin (CDDP) and then plated. Colonies were dissolved in methanol and the absorbance reads (540 nm) are represented in the bar diagrams. DMSO SB o/n

SB 3h H2O2

MDA-MB-231 Cell number

DCFDA

Figure S5. MDA-MB-231 cells were incubated for the indicated times with SB203580 (SB, 10 μM), overnight with DMSO or 10 min with H2O2 (5 mM). ROS levels were measured with DCFDA by flow cytometry

MCF7 MDA SW620 - + - + - + SB CAT SOD Tubulin

Figure S6. MCF7, MDA-MB-231 and SW620 cancer cell lines were treated overnight with SB203580 (SB, 10 μM). Total cell lysates were analyzed by immunoblotting with the indicated antibodies. CAT : ; SOD: superoxide dismutase. - SB siControlsiPTPN1siPTPN12siDUSP16siDUSP8siDUSP10

P-JNK

JNK

siControlsiPTPN1 siPTPN12siDUSP16siDUSP8siDUSP10 Tubulin P-JNK SW620

JNK

- SB siControlsiPTPN1siPTPN12siDUSP16siDUSP8siDUSP10 Tubulin P-JNK HT-29

JNK

Tubulin MDA-MB-231

Figure S7. The indicated cancer cell lines were transfected with siRNAs (50 nM) against PTPN1, PTPN12, DUSP16, DUSP8 and DUSP10, or with a scramble control, and 48 h later total cell lysates were analyzed by immunoblotting with the indicated antibodies. As a positive control, cells were incubated overnight with SB203580 (SB, 10 μM) before immunoblotting.

A B CDDP UV - - + + - - + + ASK1 inh - - 0.5 1 5 10 ASK1 inh (μM) - + - + - + - + p38 inh P-JNK p85PARP

JNK P-JNK

MCF7 JNK

P-HSP27 Tubulin MCF7 Figure S8. A. MCF7 cells were incubated with the indicated concentrations of ASK1 inhibitor (ASK1 inh) overnight and then irradiated with UV (50 J/m2) followed by 30 min incubation at 37ºC. Total cell lysates were analyzed by immunoblotting with the indicated antibodies. B. MCF7 cells were incubated overnight with SB203580 (p38 inh, 10 μM) and/or ASK1 inhibitor (10 μM) followed by cisplatin (CDDP, 100 μM) for 8 h. Total cell lysates were analyzed by immunoblotting with the indicated antibodies. A B CDDP Vehicle p38 inh Vehicle p38 inh CDDP - + - + - + - + Derivatized vehicle p38 inh CDDP +p38 inh

- + - + - + - + NaCl 97 P-Hsp27 68 Oxyblot Hsp27 43 tubulin 29 kDa 80% 100% 60% 100% Oxy/Tub Tubulin

C Vehicle p38 inh CDDP CDDP + p38 inh

Carcinoma Adenoma Adenoma Adenoma Hyperplasia Adenoma Hyperplasia H&E Ki67

82% 52% 42% 38% 44% 37% 30%

Figure S9. A. Breast tumors obtained from different treatments were incubated, immediatly after resection, for 15 min in a solution of NaCl (300 mM) and then snap frozen. Total tissue lysates were analyzed by immunoblotting with the indicated antibodies. B. Total lysates from breast tumors at day 7 were analyzed by immunoblotting using Oxyblot. The total protein oxidation signal per lane was quantified with tubulin as a reference. Results were confirmed using three mice per condition. C. H&E and Ki67 staining of breast tumors analyzed at day 7. Quantifications of Ki67 staining are indicated. Images are 20x A Vehicle p38 inh CDDP CDDP + p38 inh Carcinoma Carcinoma Adenoma Hyperplasia Carcinoma Hyperplasia Adenoma Hyperplasia H&E Ki67

71% 43% 49% 37% 48% 43% 30% 10%

B Carcinoma Hyperplasia Adenoma Non-transformed 100

80

60

40 Tumor stage (%) Tumor 20

0 vehicle p38 inh CDDP CDDP + p38 inh

Figure S10. A. H&E and Ki67 staining of breast tumors collected at day 18. Images shown are 20x. Quantifications of Ki67 staining are indicated. B. H&E stained sections of breast tumors collected at day 18 were analyzed under the microscope in blinded fashion. Samples were classified as carcinoma, adenoma, hyperplasia and normal tissue (non-transformed). relative tonon-treatedcells werecalculated. SB203580 (SB,10μM)for24h.Cellviability wasmeasuredusingtheMTT assay. The percentagesofviable cells MCF7, SW620andHT-29 cellsweretreatedwithincreasingconcentrationsofcisplatin(CDDP) orwithout Figure S12. Survival (%) for upto70days Then, asecondinjectionofCDDP wasadministeredto themiceandtumorgrowthwasmonitored MMTV-PyMT femalemicewithbreasttumorsofabout 200mm Figure S11. vehicle for15daysandtumorgrowthwasmonitoreduntil tumorsreachedagain200mm single-dose ofcisplatin(CDDP)followedbydailyadministration ofPH-797804(PH,10mg/Kg)or independent experiments,inwhichatleastfourmiceper conditionwereused. original sizeofeachtumorwhenthetreatmentbegan. The graphcompilestheresultsofthree Normalized tumor size 0.5 1.0 1.5 100 25 50 75 0 0 -10 0 IC50 =20μM IC50 =47μM 10 0 (CDDP+vehicle) +CDDP

20 MCF7 . Tumor sizewasmeasuredattheindicated timesandnormalizedrelativetothe 30 1 40 2 -10 50 IC50 =30μM IC50 =73μM 60 days log CDDP (μM) SW620 0 70

1 Normalized tumor size 1.0 0.5 1.5 0 0 2 10 3 involumeweretreatedwitha -10 20 (CDDP+p38 inh)+CDDP IC50 =309μM IC50 =370μM 30 0 HT-29 1 40 2 50 3 . 3 60 days CDDP+SB CDDP 70