Figure S1, related to Figure 1. Escaper p38a-expressing cancer cells repopulate the tumors (A) Scheme of the mt/mg reporter that expresses a

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1 Cancer Cell, Volume 33 Supplemental Information Targeting p38a Increases DNA Damage, Chromosome Instability, and the Anti-tumoral Response to Taxanes in Breast Cancer Cells Begoña Cánovas, Ana Igea, Alessandro A. Sartori, Roger R. Gomis, Tanya T. Paull, Michitaka Isoda, Héctor Pérez-Montoyo, Violeta Serra, Eva González-Suárez, Travis H. Stracker, and Angel R. Nebreda

2 1 Figure S1, related to Figure 1. Escaper p38a-expressing cancer cells repopulate the tumors (A) Scheme of the mt/mg reporter that expresses a loxp-flanked Tomato, which is excised upon Cre activation allowing the tracing of recombined cells by the expression of GFP. (B) Representative images of the mt/mg reporter expression in tumors from p38ad mice at the indicated days. Bar =100 µm (C) Representative workflow for the mt/mg reporter line by FACS analysis. CD326-APC antibody was used to detect epithelial cells. Rectangles indicate the gated epithelial cells. (D) Percentage of recombined (green) and not recombined (red) epithelial cells in the mammary tumor. Day 0, n=2; day 15, n=3; day 40, n=2. Barplots represent mean ± SEM.

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4 Figure S2, related to Figure 2. Characterization of epithelial cell lines derived from mammary tumors (A) Scheme of the protocol used to derive cancer cell lines from PyMT-induced mammary tumors. (B) Representative bright field image of cancer cells derived from PyMT-induced mammary tumors. Bar = 25 µm. (C) EPCAM-FITC fluorescence intensity of cancer cells derived from PyMT-induced mammary tumors and its corresponding isotype control. (D) Levels of g-h2ax were analyzed by immunoblotting in lysates from four independent sets of WT and p38ad cancer cells. Quantifications are shown in the histogram referring to the g-h2ax intensity in the corresponding WT cells, which was given the value of 1. (E) WT and p38a-deficient cancer cells were analyzed by immunoblotting using antibodies against the indicated proteins. (F) Cell cycle profile analysis of WT and p38a-deficient cancer cells. (G) Representative graph of the forward-scatter (FS) of WT and p38a-deficient cancer cells. The histogram shows the values from five independent experiments. (H) The percentages of multinuclear cells in WT and p38a-deficient cancer cells were calculated using E-cadherin and DAPI staining. Histogram shows the quantification of two independent experiments. Histograms represent mean ± SEM. 3

5 4 Figure S3, related to Figure 3. Oxidative stress does not mediate the deleterious effects of p38a deletion in mammary cancer cells (A) ROS levels detected using the DCFDA probe in WT and p38a-deficient cancer cells derived from PyMT-induced mammary tumors. Histogram shows the mean ± SEM of two independent experiments. Representative DCF intensity plot is shown including H2O2 treated WT cells as positive control. (B) Levels of 8-hydroxy-2 -deoxyguanosine (8-OHdG) in WT and p38a-deficient cancer cells. The histogram shows the median values ± SEM of three independent experiments. Representative 8-OHdG fluorescence intensity plot is shown including H2O2 treated WT cells as positive control. (C) Representative images of 8-OHdG staining in WT and p38a-deficient cancer cells. H2O2 treated cells were used as positive control. Bars = 50 µm. (D) Efficiency of anti-oxidants (AOX) was evaluated by treating cells with H2O2 in the presence or absence of AOX. ROS levels were detected by FACS using the DCFDA probe.

6 (E) Distribution of nuclear g-h2ax intensity in p38ad cells either non-treated or treated with AOX, and in WT cells. The midlines show median values. (F) DNA fiber assays were performed in p38ad cells either non-treated or treated with AOX, and in WT cells, and fork rate was determined. The midlines show median values. 5

Figure S4, related to Figure 4. p38a phosphorylates CtIP (A) Scheme indicating the CtIP sites phosphorylated by p38a in vitro, as determined by mass spectrometry analysis.

7 Figure S4, related to Figure 4. p38a phosphorylates CtIP (A) Scheme indicating the CtIP sites phosphorylated by p38a in vitro, as determined by mass spectrometry analysis. (B) Purified WT and T847A CtIP proteins were phosphorylated in vitro with purified active p38a and immunoblotted with antibodies for phospho-ser/thr, CtIP-pT847 or total CtIP. (C) Purified N-terminal CtIP was phosphorylated in vitro with p38a, and then analyzed by immunoblotting with the indicated phospho-antibodies. A representative Coomassie staining is shown. (D) WT and p38a-deficient cancer cells were treated 1 µm camptothecin (CPT) for 2 hr, and then analyzed by immunofluorescence using the CtIP-pT315 antibody. The percentages of cells with an intensity value higher than 45 (blue boxes) are indicated. Midlines represent the median values. 6

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9 Figure S5, related to Figure 6. CtIP but not ROS is involved in taxane-induced cancer cell death (A) Cancer cells derived from PyMT-induced mammary tumors were treated with increasing concentrations of cisplatin, paclitaxel, or docetaxel for 72 hr in the presence or absence of the p38a inhibitor LY Cell viability was measured using the CyQuant NF proliferation kit and dose-response curves were analyzed using GraphPad Prism. Data represent mean ± SEM. (B) Cancer cells derived from PyMT-induced mammary tumors were treated with control (NT) or CtIP shrnas, and then incubated with either paclitaxel 25 nm, docetaxel 10 nm or camptothecin 1 µm for 72 hr. CtIP levels were analyzed by immunoblotting (left panel), and cell death was measured by Annexin V/PI staining (right panel). The total percentage of Annexin V + cells is indicated in red. The experiment was repeated twice with similar results. (C) Cancer cells derived from PyMT-induced mammary tumors were infected with lentivirus expressing CtIP-T847E or the empty vector, and then treated with paclitaxel 25 nm, docetaxel 10 nm or camptothecin 500 nm for 72 hr. The total percentage of Annexin V + cells is indicated in red. The experiment was repeated twice with similar results (D) Cancer cells derived from PyMT-induced mammary tumors were treated with either paclitaxel 50 nm, docetaxel 10 nm or cisplatin 10 µm, with or without antioxidants (AOX) for 48 hr. Representative images after treatment are shown (left panel). Scale bar = 100 µm. Cell death was measured by Annexin V/PI staining (right panel). The total percentage of Annexin V + cells is indicated in red. The experiment was repeated twice with similar results. 8

10 Figure S6, related to Figure 6. MK2 inhibition does not sensitize PyMT mammary tumors to taxanes. (A) Growth curve of PyMT-induced mammary tumors in mice treated with vehicle or inhibitors for MK2 (MK2i, PF ) or p38a (p38i, PH797804). (B) Efficacy of the inhibitors was verified by immunoblotting with phospho-hsp27 antibody using lung tissue, which immediately after resection was incubated in NaCl (300 mm) for 15 min and then snap frozen. (C) Growth curve of PyMT-induced mammary tumors in mice treated with paclitaxel (upper panel) or docetaxel (lower panel) either alone or together with daily administration of the MK2 inhibitor. The same vehicle-treated animals are represented in both graphs. 9

11 (D) DNA damage was measured using g-h2ax staining. Histograms represent the g-h2ax + area in animals treated with paclitaxel (upper panel) or docetaxel (lower panel). At least four independent mammary tumors were quantified, with a minimum of 15 fields per tumor. (E) Mitotic figures were analyzed at the end of the treatment using phospho-h3 staining. Histograms show the percentage of missegregating cells in animals treated with paclitaxel (upper panel) or docetaxel (lower panel). At least three independent mammary tumors were analyzed, with at least 10 fields quantified per tumor. Data represent mean ± SEM. 10

11 Figure S7, related to Figure 7. Characterization of the PDX models, design of combination treatments, and lack of toxicity of PH797804 and taxanes.

12 11 Figure S7, related to Figure 7. Characterization of the PDX models, design of combination treatments, and lack of toxicity of PH and taxanes. (A) Representative images of sections from the human breast cancer PDXs stained as indicated. TN, Triple negative; Lum, Luminal; HE, Hematoxylin and eosin; ERa, Estrogen

13 receptor-a; PgR, Progesterone receptor; Ecad, E-cadherin; SMA, Smooth muscle actin. Bars = 50 µm. (B) Experimental design of the combination treatments. (C) Mouse weights were recorded once a week and values were normalized to the initial body weight of each mouse. Graphs show the weight evolution of two representative PDX models, using 4 mice per group for TN1 and 3 mice per group for LUM2. Data represent mean ± SEM. 12

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15 Figure S8, related to Figure 7. Effect of p38a and MK2 inhibitors combined with paclitaxel or docetaxel in the different PDX models (A - C) NOD/Scid female mice implanted with the indicated PDXs were treated with paclitaxel or docetaxel either alone or in combination with daily administration of the p38a inhibitor PH (A), the p38a inhibitor LY (B), or the MK2 inhibitor PF (C) for 21 days. The relative tumor growth for each treatment and PDX is shown. Tumor growth was followed for up to 20 days for the inhibitor alone, and up to 100 days or when one of the treatments reached twice the initial tumor size for the taxane and combination treatments. For each model, the same vehicle-treated animals are represented in all panels. n is indicated in Tables S1 and S3. Data represent mean ± SEM. 14

16 15 Table S1, related to Figure 7. Number of animals (n) of each PDX model used in Figures 7A and S8A. Numbers in brackets indicate the tumors analyzed. TN 1 TN 2 TN 3 TN 4 TN 5 Lum 1 Lum 2 Lum 3 Lum 4 Day 17/20 Day 23/ 29 Day 59 Day 79 Day 99 VEH n=13 (25) n=8 (13) n= 4 (6) n=6 (11) n=11 (19) n= 6 (11) n=6 (10) n=4 (8) n=5 (10) PH n=12 (24) n=7 (14) n=4 (6) n=7 (11) n=11 (22) n=6 (11) n=5 (10) n=5 (9) n=7 (13) DTX n=9 (18) n=7 (14) n=5 (7) n=6 (10) n=6 (12) n=7 (11) n=4 (8) n=8 (16) n=4 (8) DTX+PH n=7 (14) n=7 (14) n=5 (9) n=6 (12) n=8 (15) n=5 (10) n=5 (8) n=8 (15) n=4 (8) PACL n=7 (12) n=7 (14) n=5 (7) n=5 (9) n=4 (8) n=7 (10) n=9 (16) n=8 (16) n=3 (6) PACL+PH n=4 (8) n=8 (15) n=5 (7) n=6 (11) n=5 (10) n=7 (10) n=9 (16) n=7 (14) n=3 (6) DTX n=6 (11) n.a. n=5 (7) n=5 (8) n.a. n=5 (8) n=4 (8) n=6 (12) n.a. DTX+PH n=5 (10) n.a. n=5 (9) n=6 (12) n.a. n=3 (6) n=5 (8) n=6 (11) n.a. PACL n.a. n.a. n=5 (7) n=5 (9) n.a. n=4 (7) n=6 (10) n=3 (6) n.a. PACL+PH n.a. n.a. n=5 (7) n=6 (11) n.a. n=4 (6) n=7 (12) n=3 (6) n.a. DTX n=1 (2) n.a. n.a. n=4 (6) n.a. n=4 (6) n.a. n.a. n.a. DTX+PH n=1 (2) n.a. n.a. n=6 (12) n.a. n=2 (4) n.a. n.a. n.a. PACL n.a. n.a. n.a. n=5 (9) n.a. n=4 (7) n=6 (10) n.a. n.a. PACL+PH n.a. n.a. n.a. n=6 (11) n.a. n=4 (6) n=7 (12) n.a. n.a. DTX n.a. n.a. n.a. n=2 (3) n.a. n.a. n.a. n.a. n.a. DTX+PH n.a. n.a. n.a. n=5 (10) n.a. n.a. n.a. n.a. n.a. PACL n.a. n.a. n.a. n=3 (5) n.a. n=2 (3) n=5 (8) n.a. n.a. PACL+PH n.a. n.a. n.a. n=3 (5) n.a. n=3 (4) n=6 (10) n.a. n.a. n.a., not available (mice were sacrificed before this time point) Table S2, related to Figure 7. Number of tumors analyzed in Figures 7B and 7C g-h2ax Phospho-H3 TN 1 TN 2 TN 5 Lum 1 Lum 2 Lum 3 Vehicle Paclitaxel Paclitaxel+PH Docetaxel Docetaxel+PH Vehicle Paclitaxel Paclitaxel+PH Docetaxel Docetaxel+PH

17 16 Table S3, related to Figure 7. Number of animals (n) of each PDX model used in Figures S8B and S8C. Numbers in brackets indicate the tumors analyzed. TN 1 TN 2 Lum 1 Lum 2 Day 17/20 Day 23/ 29 Day 59 Day 79 Day 99 LY n=4 (7) n=3 (6) n=4 (8) n=3 (4) MK2i n=3 (6) n=3 (6) n=3 (5) n=3 (4) DTX+LY n=5 (10) n=4 (8) n=5 (8) n=3 (4) DTX+MK2i n=3 (6) n=4 (8) n=3 (4) n=3 (4) PACL+LY n=3 (6) n=4 (8) n=3 (6) n=3 (6) PACL+MK2i n=3 (6) n=4 (7) n=3 (5) n=3 (5) DTX+LY n=5 (10) n.a. n=3 (4) n=3 (4) DTX+MK2i n=3 (6) n.a. n=3 (4) n=3 (4) PACL+LY n.a. n.a. n=3 (6) n=3 (6) PACL+MK2i n.a. n.a. n=3 (5) n=3 (5) DTX+LY n=5 (10) n.a. n=4 (6) n.a. DTX+MK2i n=2 (4) n.a. n=2 (4) n.a. PACL+LY n.a. n.a. n=3 (6) n.a. PACL+MK2i n.a. n.a. n=3 (5) n.a. DTX+LY n.a. n.a. n.a. n.a. DTX+MK2i n.a. n.a. n.a. n.a. PACL+LY n.a. n.a. n=3 (6) n.a. PACL+MK2i n.a. n.a. n=2 (3) n.a. n.a., not available (mice were sacrificed before this time point) Table S4, related to Figure 8. Number of tumors analyzed in Figures 8A-8C TN 1 TN 2 TN 3 TN 4 TN 5 Lum 1 Lum 2 Lum 3 Lum 4 Aneuploidy rate g-h2ax + area Missegregation rate

SHREE ET AL, SUPPLEMENTAL MATERIALS. (A) Workflow for tumor cell line derivation and orthotopic implantation.

SHREE ET AL, SUPPLEMENTAL MATERIALS SUPPLEMENTAL FIGURE AND TABLE LEGENDS Supplemental Figure 1. Derivation and characterization of TS1-TGL and TS2-TGL PyMT cell lines and development of an orthotopic