Supplemental Methods. Cell culture and generation of stable cell lines. Primary mouse embryonic fibroblasts (MEFs) were isolated from E13.5 embryos derived.
Merkwirth_et al. Supplemental Data
Prohibitins control cell proliferation and apoptosis by regulating OPA1dependent cristae morphogenesis in mitochondria
Carsten Merkwirth, Sascha Dargazanli, Takashi Tatsuta, Stefan Geimer, Beatrix Löwer, F. Thomas Wunderlich, Jürgen-Christoph von Kleist-Retzow, Ari Waisman, Benedikt Westermann, and Thomas Langer
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Merkwirth_et al. Supplemental Methods Cell culture and generation of stable cell lines Primary mouse embryonic fibroblasts (MEFs) were isolated from E13.5 embryos derived from intercrosses of time-mated pregnant Phb2fl/+ mice. Immortalization of early passage primary MEFs was achieved by SV40 transformation (Todaro and Green 1965). MEFs were cultured in Dulbecco´s Modified Eagle´s Medium (DMEM) (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Invitrogen), 100 U/ml penicillin (PAA), 100 µg/ml streptomycin (PAA), 100 µM non-essential amino acids (PAA), 2 mM L-glutamine (PAA) and 1 mM sodium pyruvate (PAA). All cells were cultured at 37°C in an atmosphere of 5% CO2 and 90% humidity. For the generation of stable cell lines 1x107 SV40-transformed Phb2fl/fl MEFs were transfected via electroporation with 40 µg of the pCAGs-STOP-IRES-EGFP plasmid containing different murine Phb2 cDNAs. 24 hrs after transfection cells were selected with 300 µg/ml G418 (PAA) for 9 days. Single G418-resistant MEF clones were isolated and expanded for further analysis. Stable expression of the transgenes was examined via immunoblotting.
RT-PCR RNA was isolated from MEFs using the RNeasy mini kit (Qiagen) according to the manufacturer´s instructions. cDNA synthesis was performed with the Transcriptor cDNA synthesis kit (Roche) following the manufacturer´s guidelines.
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Merkwirth_et al. Respiratory chain function Oxygen consumption studies of viable cells and spectrophotometric evaluation of respiratory chain and TCA cycle enzyme activities in frozen homogenates were performed as previously described (Rustin et al. 1994). Mitochondria were isolated from control and PHB2-deficient MEFs according to described methods (Fernandez-Vizarra et al. 2002), separated on 12% SDS-PAGE and electroblotted on nitrocellulose membranes (Schleicher & Schuell). Steadystate levels of respiratory chains subunits were determined via immunoblotting using the following antibodies: mouse anti-complex II, 70 kDa (Molecular Probes, 1:1000), mouse anticomplex I, 39 kDa (Molecular Probes, 1:1000), mouse anti-complex V, subunit a (Molecular Probes, 1:1000), mouse anti-complex III, subunit IIa (Molecular Probes, 1:1000), mouse antiporin/VDAC (Calbiochem, 1:1000), mouse anti-Tim23 (BD Biosciences, 1:500), rabbit antiAconitase (1:1000), mouse anti-Hsp60 (Stressgen, 1:1000).
Immunoblotting MEFs were harvested from tissue culture plates either by trypsinization or by cell scraping and lysed in protein cell lysis buffer (10 mM Tris-Cl pH7.4, 10 mM EDTA, 50 mM NaCl, 50 mM NaF, 1% Triton X-100 supplemented with 20 µg/ml aprotinin, 2 mM sodiumorthovanadate, 1 mM PMSF and Complete Mini Protease inhibitor cocktail mix) for 2 hrs at 1500 rpm in a Vibrax shaker. Protein concentrations were determined with a standard Bradford protein assay (BioRad). 50-200 µg of total protein were separated on SDS-PAGE, transferred to nitrocellulose membranes (Schleicher & Schuell) and subjected to immunoblotting with the following antibodies: rabbit anti-BAP37 (BioLegend, 1:500), rabbit anti-prohibitin (NeoMarkers, 1:500), mouse anti-OPA1 (BD Biosciences, 1:500), rabbit antiPARP (Cell Signaling, 1:1000), rabbit anti-cleaved caspase-3 (Cell Signaling, 1:1000), rabbit
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Merkwirth_et al. anti-Bax (Santa Cruz, 1:1000), rabbit anti-Bcl2 (Santa Cruz, 1:200), rabbit anti-p53 (SantaCruz, 1:200), rabbit anti-phospho-p53 (Genetex, 1:1000), mouse anti-Hax1 (BD Biosciences, 1:500), mouse anti-FLAG M2 (Sigma, 1:1000) and mouse anti-β-actin (Sigma, clone AC15, 1:5000).
Flow cytometry Mitochondrial membrane potential was measured by fluorescence-activated cell sorting after staining MEFs of the indicated genotypes with JC-1 or TMRM dyes (Molecular Probes) as recommended by the manufacturer. To determine cell proliferation by flow cytometry, 5x105 MEFs transduced with Crerecombinase were labelled with 0,5 µM carboxyfluorescein diacetate succinimidyl ester (CFSE) (Asquith et al. 2006) and subjected to FACS analysis.
Supplementary References Asquith, B., Debacq, C., Florins, A., Gillet, N., Sanchez-Alcaraz, T., Mosley, A., and Willems, L. 2006. Quantifying lymphocyte kinetics in vivo using carboxyfluorescein diacetate succinimidyl ester (CFSE). Proc R Soc B 273: 1165-1171. Fernandez-Vizarra, E., Lopez-Perez, M.J., and Enriquez, J.A. 2002. Isolation of biogenetically competent mitochondria from mammalian tissues and cultured cells. Methods 26(4): 292-297. Rustin, P., Chretien, D., Bourgeron, T., Gerard, B., Rotig, A., Saudubray, J.M., and Munnich, A. 1994. Biochemical and molecular investigations in respiratory chain deficiencies. Clin Chim Acta 228(1): 35-51. Todaro, G.J. and Green, H. 1965. Successive Transformations Of An Established Cell Line By Polyoma Virus And Sv40. Science 147: 513-514.
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Merkwirth_et al. Legends to Supplemental Table 1 Table S1: Embryonic lethality of Phb2 ablation in mice (A) Embryonic lethality of PHB2-deficient mice. Analysis of progeny derived from intercrossings of Phb2+/- mice. Embryos from the indicated stages or newborn mice were genotyped by PCR using Phb2 allele-specific primers. Expected numbers reflect the mendelian ratio. E, embryonic day (B) Brain-specific inactivation of murine Phb2 causes embryonic lethality . Analysis of progeny derived from breedings of Nestin-Cre/Phb2fl/+ males to Phb2fl/fl females . Newborn animals were genotyped by PCR for Phb2 and the Nestin-Cre transgene. Expected numbers reflect the mendelian ratio. Nes-Cre refers to Nestin-Cre.
Legends to Supplemental Figures
Figure S1. Homologous recombination of the Phb2 gene replacement vector in murine ES cells Southern blot analysis of wild type and ES cell clones harbouring the targeted Phb2fl(neo)/+ allele after homologous recombination. Genomic DNA isolated from wild type (WT) and Phb2fl(neo)/+ ES cells (targeted) was digested with HindIII, hybridized with an external 5´ probe (Probe A) and an external 3´ probe (Probe B), and analyzed by autoradiography.
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Merkwirth_et al. Figure S2. Phb2 deletion does not affect the steady state level of pro- and anti-apoptotic proteins (A) Immunoblot analysis of MEF lysates using the indicated antibodies. β-actin-specific antibodies were used as a loading control. p-p53, phosphorylated p53. (B) Increased loss of viability of prohibitin-deficient MEFs upon induction of apoptosis. Cretransduced Phb2fl/fl MEFs were treated with different concentrations of H2O2 for 4 hrs and stained with annexin-V and propidium iodide. Viability was determined cytofluorimetrically as the percentage of annexin-V-/propidium iodide-negative cells. Phb2fl/fl untreated cells were set to 100% viability. * ρ
