German Research Center for Environmental Health GmbH, Munich, Germany

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1 PPAR is Necessary for Radiation-Induced Activation of Non-Canonical TGF signaling in the Heart Vikram Subramanian 1, Sabine Borchard 2, Omid Azimzadeh 1, Wolfgang Sievert 3, Juliane Merl-Pham 4, Mariateresa Mancuso 5, Emanuela Pasquali 5, Gabriele Multhoff 3, Bastian Popper 6, Hans Zischka 2,7, Michael J. Atkinson 1,8, Soile Tapio 1* 1 Institute of Radiation Biology, Helmholtz Zentrum München - German Research Center for Environmental Health GmbH, Munich, Germany 2 Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum München - German Research Center for Environmental Health GmbH, Munich, Germany 3 Department of Radiation Oncology, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany and Institute of innovative Radiotherapy (irt), Helmholtz Zentrum München, German Research Center for Environmental Health GmbH Munich, Germany 4 Research Unit Protein Science, Helmholtz Zentrum München - German Research Centre for Environmental Health, Munich, Germany 5 Laboratory of Radiation Biology and Biomedicine, Agenzia Nazionale per le Nuove Tecnologie, l Energia e lo Sviluppo Economico Sostenibile (ENEA), Rome, Italy 5 Department of Cell Biology and Core Facility Animal Models (CAM), Biomedical Center, Ludwig-Maximilians University Munich, Planegg, Germany 7 Institute of Toxicology and Environmental Hygiene, Technical University of Munich, Munich, Germany S 1

2 8 Chair of Radiation Biology, Technical University of Munich, Munich, Germany Figure S-1. Genotyping of wild type, PPARα heterozygous, and PPARα homozygous mutant mice. Figure S-2. Typical electron microscopy images of heart sections of the non-irradiated wild type mice (a), wild type mice irradiated at 8 Gy (b) or 16 Gy (c), non-irradiated PPAR heterozygous mutant mice (d), heterozygous mutant mice irradiated at 8 Gy (e) or 16 Gy (f), and non-irradiated PPAR homozygous mutant mice (g), homozygous mutant mice irradiated at 8 Gy (h) or 16 Gy (i). Figure S-3. Immunoblot analysis of phosphorylated and total levels of DRP1 in wild type (W), PPARα heterozygous (H) mutant mice and PPARα homozygous (M) mutant mice. Figure S-4. Immunoblot analysis of total levels of PGC1 in wild type (W), PPARα heterozygous (H) mutant mice and PPARα homozygous (M) mutant mice. Figure S-5. Immunoblot analysis of phosphorylated and total levels PPARα and PPAR are shown in wild type (W) and heterozygous (H) mice in control (0 Gy) and irradiated (8 and 16 Gy) animals. Figure S-6. Immunoblot analysis of MMP9 and SMAD 4 expression in wild type (W), PPARα heterozygous (H) mutant mice and PPARα homozygous (M) mutant mice. Figure S-7. Immunoblot analysis of phosphorylated and total levels TAK1, JNK2, c-jun in wild type (W), PPARα heterozygous (H) mutant mice and PPARα homozygous (M) mutant mice. S 2

3 Figure S-8. Immunoblot analysis of expression of α-sma, vimentin, vinculin and paxillin is shown in wild type (W), PPARα heterozygous (H) mutant mice and PPARα homozygous (M) mutant mice. Table S-1. All proteins identified by label free proteomics. Table S-2. All proteins quantified by label free proteomics. Table S-3. Significantly deregulated proteins in irradiated heart after 8 Gy in PPARα wild type. Table S-4. Significantly deregulated proteins in irradiated heart after 16 Gy in PPARα wild type. Table S-5. Significantly deregulated proteins in irradiated heart after 8 Gy in PPARα +/-. Table S-6. Significantly deregulated proteins in irradiated heart after 16 Gy in PPARα +/-. Table S-7. Significantly deregulated proteins in irradiated heart after 8 Gy in PPARα -/-. Table S-8. Significantly deregulated proteins in irradiated heart at 16 Gy in PPARα -/-. Table S-9. Significantly deregulated proteins in sham-irradiated PPARα +/- compared to the sham-irradiated PPARα +/+ (wild type). Table S-10. Significantly deregulated proteins in sham-irradiated PPARα -/- compared to the sham-irradiated PPARα +/+ (wild type). S 3

4 1000 bp 900 bp 800 bp 700 bp 600 bp 500 bp 400 bp 300 bp 200 bp 100 bp M +/ / +/+ N Figure S-1. Genotyping of wild type, PPARα heterozygous, and PPARα homozygous mutant mice. Genotyping was performed by polymerase chain reaction. Genomic DNA produced a band of 412 bp in wild type, a mutant band of 650 bp in PPARα -/- and both wild type and mutant band in PPARα +/- mice. M = 100bp marker, +/+ = wild type mice, +/- = PPARα +/- mice, -/- = PPARα -/- mice, N=negative control S 4

5 Figure S-2. Typical electron microscopy images of heart sections of the nonirradiated wild type mice (a), wild type irradiated mice at 8 Gy (b) or 16 Gy (c), non-irradiated PPAR heterozygous mutant mice (d), heterozygous mutant mice irradiated at 8 Gy (e) or 16 Gy (f), and non-irradiated PPAR homozygous mutant mice (g), homozygous mutant mice irradiated at 8 Gy (h) or 16 Gy (i). Magnification 2,000X. S 5

6 Figure S-3. Immunoblot analysis of phosphorylated and total levels of DRP1 in wild type (W), PPARα heterozygous (H) mutant mice and PPARα homozygous (M) mutant mice. The radiation doses are indicated. n = 4 S 6

7 Figure S-4. Immunoblot analysis of total levels of PGC1 in wild type (W), PPARα heterozygous (H) mutant mice and PPARα homozygous (M) mutant mice. The radiation doses are indicated. (2-way ANOVA with Bonferroni post-hoc test; * p < 0.05, ** p < 0.01; n = 4). S 7

8 Gy 8 Gy 16 Gy W H M Figure S-5. Immunoblot analysis of phosphorylated and total levels PPARα and PPAR are shown in wild type (W) and heterozygous (H) mice in control (0 Gy) and irradiated (8 and 16 Gy) animals. The radiation doses are indicated. Columns represent the average ratios of relative protein expression of phosphorylated (Ser112) S 8

9 PPAR in control and irradiated samples after background correction and normalization to Ponceau. The error bars are calculated as SD. (t-test; *p 0.05; n = 4). Figure S-6. Immunoblot analysis of MMP9 and SMAD 4 expression in wild type (W), PPARα heterozygous (H) mutant mice and PPARα homozygous (M) mutant mice. The radiation doses are indicated. n = 4 S 9

10 Figure S-7. Immunoblot analysis of phosphorylated and total levels TAK1, JNK2, c-jun in wild type (W), PPARα heterozygous (H) mutant mice and PPARα homozygous (M) mutant mice. The radiation doses are indicated. n = 4 Figure S-8. Immunoblot analysis of expression of α-sma, vimentin, vinculin and paxillin is shown in wild type (W), PPARα heterozygous (H) mutant mice and PPARα homozygous (M) mutant mice. The radiation doses are indicated. n = 4 S 10