DI LISA, Fabio

Mongillo, Marco

R., Menab\uf2

R., Quintana Cabrera

Sandri, Marco

Scorrano, Luca

SORIANO GARCIA - CUERVA, MARIA EUGENIA

T., Varanita

T., Zaglia

V., Costa

V., Romanello

English

T. Varanita

R. Quintana Cabrera

V. Romanello

T. Zaglia

R. Menabò

V. Costa

DI LISA, FABIO

MONGILLO, MARCO

SANDRI, MARCO

SCORRANO, LUCA

Archivio istituzionale della ricerca - Università di Padova

 Controlled overexpression of the mitochondria shaping protein Optic Atrophy 1 counteracts cellular damage in vivo Tatiana Varanita, Maria Eugenia Soriano, Ruben Quintana Cabrera, Vanina Romanello, Tania Zaglia, Roberta Menabò, Veronica Costa, Fabio Di Lisa,  Marco Mongillo, Marco Sandri, and Luca Scorrano  Supplementary online material   Legends to supplementary figures Supplementary Figure 1S. Characterization of Opa1tg mice (A) Equal amounts of protein from liver tissue of the indicated genotypes were separated by SDS-PAGE and immunoblotted with the indicated antibodies.  (B) Equal amounts of protein from liver tissue of the indicated genotypes were separated by SDS-PAGE and immunoblotted with the indicated antibodies.  (C) Growth rate of  C57/Bl6 Wt and Opa1tg males and females during the initial 60 days of life. Data  represent  average + SEM (n=20 per each group). (D) Body weight is represented as average + SEM of 5 months males (n=19 for each group) and females (n= 12 for each group) and 9 months old males (n= 32 for each group) and females (n=18 for each group) SV129 Opa1tg  and Wt litettermates.    Supplementary Figure 2S. Histological and morphological characterization of heart, liver and kidney (A) Immunofluorescence analysis on ventricular cryosections from Wt and Opa1tg  mice stained with an antibody to dystrophin. Images are details from the left ventricle. Scale bars, 25 µm.  (B) Evaluation of cardiomyocyte (CM) area in cryosections from Wt and Opa1tg 5 months old hearts. Bars indicate average + SEM. (n=5 for each group).  (C) Echocardiographic long axis view of hearts from  5 months old Wt and Opa1tg littermates. LV: left ventricle; A: aorta.  (D) Haematoxilin-eosin staining in ventricular cryosections from 5 months Wt and Opa1tg  mice. Scale bars, 100 µm. (E) Immunofluorescence analysis of heart cryosections from 9 months old  Wt and Opa1tg mice stained with an antibody to Collagene I (Col.I).  (F) Evaluation of liver weight/body weight ratio in  5 and 9 months old male mice. Wt and Opa1tg . Bars indicate average + SEM (n=10 for each group). (G) Echographic view of the liver from 9 months old Wt and Opa1tg mice.  (H) Echographic short axis view of the left kidney from  9 months old Wt and Opa1tg mice.  (I) Morphometric analysis of kidney from 9 months old Wt and Opa1tg mice. Error bars indicate SEM (n=6 for each group).    Suplimentary Figure 3S. Autophagic program is not altered in Opa1tg mice (A) mRNAs expression of atrophy-related and muscle specific genes in Opa1tg and Wt littermates, control and denervated muscle (n=5 for each group). Data are average ±SEM. The asterisk denotes p<0.05 in an unpaired sample Student’s test. (B) mRNAs expression of autophagic genes  in Opa1tg and Wt littermates, control and denervated muscle (n=5 for each group). Data are average ±SEM. The asterisk denotes p<0.05 in an unpaired sample Student’s test. (C) mRNAs expression of mitochondrial genes  in Opa1tg and Wt littermates, control and denervated muscle (n=5 for each group). Data are average ±SEM. The asterisk denotes p<0.05 in an unpaired sample Student’s test. (D) The time course of LC3 II levels  in  mouse adult fibroblasts (MAFs) of the indicated genotypes upon autophagy induction by serum deprivation. Actin is shown as a loading control.  Densitometric analysis of LC3 II over actin. Data represent average + SEM (n=5 independent experiments). (B) The time course of LC3 II levels  when cells were deprived of serum in the presence of        100 µM chloroquine (CQ). Actin is shown as a loading control. Densitometric analysis of LC3 II over actin. Data represent average + SEM (n=5 independent experiments).      Suplimentary Figure 4S. Opa1tg mitochondria are slightly elongated and contain tighter cristae (A) Complex IV dependent respiration of mitochondria isolated from livers of the indicated genotypes. Data represent  average + SEM (n=4 for each group).  (B) Representative traces of Calcium Retention Capacity (CRC) of liver mitochondria isolated from 5 months old Wt and Opa1tg mice. For the assessment of the maximal CRC, 2 µM Cyclosporin A (CsA) was present in the medium.  (C) Quantification of CRC in experiments as in (D).  (D) Representative traces of Rhodamine 123 fluorescence. Mitochondria isolated from livers of the indicated genotype (1 mg/mL, MLM) were treated where indicated (arrows) with 300 µM ADP and 200 nM FCCP.  (E) Mitochondria of the indicated genotype were treated with cBID for the indicated times. 10 mM BMH or DMSO was then added and after 30 min the crosslinking reaction was quenched. Equal amounts (40µg) of mitochondrial proteins were analyzed by SDS-PAGE/immunoblotting using anti-BAK antibody. Asterisks indicate BAK multimers.   B10 20 30 40 50 60 704681012141618202224Weight(gr)Days after birth  Wt male  Opa1tg male Wt female Opa1tg femaleC DAWttgOpa1Mfn1Mfn2DRP1b-tubWttgOpa15mo.Males5mo.Female9mo.Males9mo.Female05101520253035  Wt  Opa1tgBodyweight(g)GRP75BCL-XLBCL-2BAXA1BAKCyto c0,010,020,030,040,050,060,079 mLiver/Bodyweight  Wt Opa1tg5 mWtOpa1tg50100150200250300350  Wt Opa1tgCMarea(mm2 )024681012  Wt Opa1tgTrasv LL LongTrasv APDistance(mm)WttgOpa1 tgOpa1Col. ICol. IWtWttgOpa1WttgOpa1A B C D EF G H IWttgOpa1RV    IVS       LVRV    IVS       LVLC3ILC3II actin  0   1  4   0  1  4 hrsWt tgOpa1Starv. + CQLC3ILC3II actin  0   1  4   0  1  4 hrsWt tgOpa1Starv.0h 1h 4h0,000,010,020,030,040,050,060,070,080,090,100,110,12  Wt Opa1tgLC3II/actinproteinlevel(a.u.)0h 1h 4h0,00,10,20,30,40,50,60,70,80,91,01,1LC3II/actinproteinlevel(a.u.)  Wt Opa1tgOpa1ConOpa1DenMfn2ConMfn2DenDrp1ConDrp1DenFis1ConFis1DenPgc1aConPgc1aDen0,00,51,01,52,02,53,0*  Wt Opa1tg*Mitochondrialgenes(foldinduction)LC3conLC3denp62conp62denBnip3conBnip3denMul1conMul1denCathLconCathLden012345678*  Wt Opa1tg*Autophagicgenes(foldinduction)FoxO1conFoxO1denFoxO3conFoxO3denFoxO4conFoxO4denActRIIBconActRIIBdenAIk3conAIk3denMyostatinconMyostatinden0123456 Wt Opa1tgAtrophyrelatedgenes(foldinduction)*A B CD E20406080100120140 Wt  Opa1tgnmolO2/min/ml         Complex IVdependent respiration0 240 480 720 960 12002404807209601200CalciumGreen-5Nfluorescence  Wt+CsA Wt Opa1tg Opa1tg+CsAtime (sec) Basal Maximal20406080100120140160180200220240260CRC,uMCa2+ Wt Opa1tgA B0 240 480 720 9601200200250300350400450500550600650ADPMito  Wt Opa1tgRhodamine123fluorescencetime (sec)FCCPDMSOWt tgOpa1cBID     0’15’30’ 0’15’30’66-46-30-21- BAKC DE BMHWt tgOpa1BAK*cBID     0’15’30’ 0’15’30’66-46-30-21-F

Controlled overexpression of the mitochondria shaping protein Optic Atrophy 1 counteracts cellular damage in vivo

https://www.research.unipd.it/bitstream/11577/3157494/1/Varanita%20et%20al%20supplementary.pdf

Controlled overexpression of the mitochondria shaping protein Optic Atrophy 1 counteracts cellular damage in vivo

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Archivio istituzionale della ricerca - Università di Padova