Supporting InformationDin et al. 10.1073/pnas.1213294110SI MethodsAdenoviral Constructs and Transgenic Mice. Generation of adEGFP,EGFP-Pim-1-dominant negative (adEGFP-PimDN), and adEGFP-PimWTadenoviral constructs,K79Mkinase-deadPimDN-transgenicmice were reported previously (1–3).

NRCM Cell Culture and Adenoviral Infection.Neonatal rat ventricularmyocyte (NRCM) maintenance and transduction were performedas previously described (1, 3).

Simulated Ischemia/Reperfusion. After adenoviral infection, simu-lated ischemia (sI) and/or sI reperfusion (sI/R) treatments weredone in glucose-free media with the following components: 1 Ldouble deionized water, 3.7 g sodium bicarbonate, 1 vial glucose-free DMEM powder (Sigma D5030), 80 μL 10 mg/mL Fungizone(Invitrogen), and 1% penicillin-streptomycin-glutamine (PSG),followed by sterile filter with a pH of 7.4. To glucose-free media,5 mM sodium cyanide and 20 mM 2-deoxy-glucose were added,followed by incubation of cells for indicated times. For sI/R, 10mM glucose was added after sI treatment for indicated times.

Plasmids and Transfections. Drp1-S637A point mutant construct waskindly provided by L. Scorrano (Venetian Institute of MolecularMedicine,Padova, Italy).CotransfectionsweredonewithDrp1-S637A,mCherry plasmid (Clontech) or Pim-1-mCherry. Transfectionswere done with Xtremegene (Roche) at a ratio of 1:3 DNA toXtremegene in 10-cm dishes of 1.6 × 105 HeLa cells, according tomanufacturer’s instructions. HeLa cells were maintained in 10%FBS DMEM.

Mitochondria Morphological Analysis and Proximity Ligation Assay.After indicated sI or sI/R treatments, cells were washed twice withPBS and fixed in 4% paraformaldehyde. Cells were then stainedwith Tom20 (Santa Cruz, 1:200). Mitochondria were imaged byconfocal microscopy on a Leica TCS SP2 laser scanning spectralconfocal microscope. The extent of mitochondrial fragmentationwas qualified on a cell-to-cell basis and scored as reticular orfragmented. Proximity ligation assay was performed according tothe manufacturer’s protocol (Olink Biosciences), using Drp1antibody (1:50, BD Transduction Laboratories), Pim-1 antibody(1:50, Cell Signaling Technologies), and p53 up-regulated mod-ulator of apoptosis (PUMA) antibody (1:50, Abcam), followed byvisualization by confocal microscopy.

Subcellular Fractionation and Immunoprecipitation. Subcellular frac-tionation of cultured cells and isolated heart tissue was performedas described previously (1). Immunoprecipitation was performedon isolated cells in 70 mM sucrose, 190 mM mannitol, 20 mMHepes solution, and 0.2 mM EDTA solution. Cells were dounced20 times, and 10 μL lysate was taken as input, followed by cen-trifugation at 10,000 × g for 10 min. Supernatant was collectedand cleared with IgG beads (Santa Cruz) for 2 h, and the beadswere removed by centrifugation for 30 s at 800 × g. One micro-gram antibody Drp1 (BD Transduction Laboratories), Pim-1(Invitrogen), PUMA (Abcam), and β-actin (Santa Cruz) was in-cubated with the sample overnight. IgG beads were added to

mixture the next day and incubated for 2 h, followed by centri-fugation of antibody IgG complex for 30 s at 800 × g. Complexwas washed twice with wash buffer (40 mM Hepes at pH 7.4, 150mM NaCl, 2 mM EDTA, 10 mM pyrophosphate, 10 mM glycero-phosphate, and 0.3% CHAPS), followed by the addition of 25 μL1× SDS sample buffer for immunoblot.

Cycloheximide Inhibition. Protein half-life was determined usingadenovirally infected NRCMs treated with cycloheximide (100 μg/mL) to inhibit protein synthesis. After the indicated time points,NRCMs were isolated in 1× SDS sample buffer and immunoblotswere performed. Proteasome inhibition was accomplished usingMG-132 at a concentration of 10 μM in the presence of cyclo-heximide for the indicated time points, followed by sample col-lection in sample buffer.

Ischemia/Reperfusion Surgery. I/R surgery was performed as pre-viously described, with some modifications. Briefly, sex-matchedFVB mice 12–14 wk of age were mechanically ventilated throughan endotracheal cannula and anesthetized until areflexic witha 1.6% isoflurane/oxygen mixture. Thermal regulation was main-tained at 37°C with a homoeothermic blanket and probe. A leftthoracotomy through the fourth intercostal space was performedto allow access to the heart, and the left anterior descending artery(LAD) was reversibly ligated (slip knot) with a 7.0 silk suture tiedtransversely around a 1–2-mm piece of PE-10 tubing 1 mm belowthe atrial apex. The thoracic opening was closed and the mice wereallowed to revive under 100% air ventilation and kept warm ona heating blanket for 50 min. After infarction, mice were re-intubated and anesthetized as before, the thoracic cavity was re-opened, the slipknot was loosened, and the tubing was removed toallow reperfusion. The thoracic cavity was securely closed and theanimal allowed to recover on a warming blanket.

In Vitro Kinase Assay. His-Pim-1 (wild-type and kinase dead) weregenerated as previously described (4). Recombinant Drp1 proteinwas purchased from Abnova. Half a microgram Drp1 was in-cubated with 10 μCi γ-32P-ATP (PerkinElmer Life Sciences), and0.5 μg Pim-1 kinase (wild-type or kinase dead) in 30 μL kinasebuffer containing 25 mM Hepes at pH 7.5, 10 mM MgCl2 for20 min at room temperature. The reaction was stopped by theaddition of 2 × SDS sample buffer and boiling for 5 min. Sampleswere separated by 4–12% SDS-PAGE, and Drp1 phosphoryla-tion status by Pim-1 was visualized by autoradiography.

Cell Death Assay. NRCMs were infected with the indicated ade-noviruses both with and without sI. Scrambled siRNA (LifeTechnologies) and Drp1 siRNA (Life Technologies) were trans-fected into NRCMs using HiPerfect (Qiagen) in minimal media,and knockdown was achieved 48 h later. Cell death assay was doneby propidium iodide staining (Molecular Probes) on NRCMs.Briefly, after sI treatment, media was collected, NRCMs weretrypsinized with 0.05% trypsin, and cells were incubated in 1 ×PBS containing 10% FBS. Cells were spun down at 1800 × g for5 min and resuspended in propidium iodide (PI) to label deadcells, immediately followed by FACS analysis.

1. Borillo GA, et al. (2010) Pim-1 kinase protects mitochondrial integrity in cardiomyocytes.Circ Res 106(7):1265–1274.

2. Muraski JA, et al. (2008) Pim-1 kinase antagonizes aspects of myocardial hypertrophyand compensation to pathological pressure overload. Proc Natl Acad Sci USA 105(37):13889–13894.

3. Muraski JA, et al. (2007) Pim-1 regulates cardiomyocyte survival downstream of Akt.Nat Med 13(12):1467–1475.

4. Zhang Y, Wang Z, Li X, Magnuson NS (2008) Pim kinase-dependent inhibition of c-Mycdegradation. Oncogene 27(35):4809–4819.

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Fig. S1. Drp1 inhibition by mdivi-1. (A) Representative confocal image of NRCMs treated with 50 μMmdivi-1 labeled with Tom20. (B) Analysis of mitochondrialmorphology in NRCMs treated with full-media, glucose-free , sI for 30 min with mdivi-1 or DMSO control. +D, DMSO; FM, full-media; GF, glucose-free; +I, mdivi-1; **, significant compared with FM; #, significant compared with sI+D. (C) TUNEL analysis between mdivi-1- and DMSO-treated cells after sI/R (30 min/20 h).mdivi-1 and DMSO were present during all times of treatment. ***, significant compared to vehicle sI/R. (D) Mice were injected i.p. with mdivi-1 or DMSOvehicle and subjected to LAD ligation–induced I/R (50 min/24 h). The hearts were excised, labeled with phthalocyanine-blue dye, and sectioned. Incubating in1% TTC was used to identify viable tissue and phthalo-blue to delineate the area at risk. Representative images and a graph of 1-mm heart sections stainedwith TTC and phthalocyanine-blue are shown. (E) Measurement of infarct size in mdivi-1- and DMSO-treated groups. **, significant compared with DMSOcontrols. (F) No significant difference in area at risk was noted between the DMSO and mdivi-1 groups. #P < 0.05; *P < 0.05; **P < 0.01; ***P < 0.001.

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Fig. S2. Pim-1 mediates Drp1 localization and protein levels. (A) Immunoblot and analysis of total Drp1 expression against total VDAC in NRCMs infected withadEGFP or EGFP-Pim-1 (adPimWT) adenovirus and subjected to sI. (B) Total Drp1 protein expression normalized to total VDAC levels in NRCMs infected with adEGFPof adPDN adenovirus. *, significant compared with EGFP. (C and D) Drp1 localization in adEGFP- and adPim-WT- or adEGFP- and adPDN-infected, fractionatedNRCMs after sI. (E) Phosphorylation status of Drp1-S637 in adEGFP-, adPim-WT-, and adPDN-infected NRCMs. *, is significant compared with EGFP control. *P < 0.05,**P < 0.01.

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Fig. S3. Drp1 half-life is not altered with Pim-1. (A) Cycloheximide chase was performed on adEGFP- and adPimWT-infected NRCMs followed by analysis ofproteosomal degradation of Drp1 with MG132. CHX, cycloheximide.

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Fig. S4. Pim-1 maintains reticular mitochondrial morphology in response to sI. (A) Confocal images displaying mitochondrial morphology of NRCMs trans-duced with adEGFP, adPimWT, and adPDN adenovirus and subjected to 30 min sI. (B) Quantitation of reticular mitochondrial morphology in NRCMs with siRNAagainst Drp1 and rescued with PimWT and or EGFP control adenovirus. *, significant compared with respective scrambled control; #, significant compared withEGFP scrambled (SCR) with sI. All sI conditions had significantly less reticular mitochondria compared with their respective controls. (C) Immunoblot confirmingdown-regulation of Drp1 followed by siRNA Drp1. **P < 0.01; ###P < 0.001; ***P < 0.001.

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Fig. S5. Pim-1 does not rescue Drp1-S637A-mediated mitochondrial fragmentation and localization. (A) Confocal images displaying mitochondrial morphologyof cardiac fibroblast transfected with control plasmid expressing mCherry, Drp1-S637A, or Pim-1-mCherry plasmid. (B) Immunoblot demonstrating Drp1-S637A

localization with and without Pim-1-mCherry.

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Fig. S6. Drp1 binds to PUMA in vitro. (A) Proximity ligation assay demonstrating endogenous interaction of PUMA and Drp1 in NRCMs. (B) Proximity ligationassay was performed with adPumaDN NRCMs. Red dots display a single interaction between Drp1 and PumaDN under baseline conditions (C) NRCMs wereinfected with adPimWT, adDrp1, or adPumaDN, as indicated (Upper). Drp1 and PimWT were immunoprecipitated with specific antibodies indicated beneaththe immunoblot. Immunoprecipitated complexes were stained for GFP to detect adPimWT and adPumaDN or Drp1, as indicated (Left). Red boxes highlightevidence for PumaDN and Drp1 interaction.

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