SIRT3 and SOD2 maintain osteoblast differentiation and bone formation by regulating mitochondrial stress

Supplementary Methods

BN-PAGE analysis of mitochondrial supercomplex

Crude mitochondrial extracts were isolated from osteoblasts under different treatment

as described previously1. Blue native electrophoresis (BN-PAGE) was performed in 4-

16% gradient gels following previously published method with minor modifcation2.

Briefly, 100 μg isolated mitochondria were solubilized with 6 g digitonin per g

mitochondrial protein. The extracts were centrifuged at 4 for 15 min at 15,000 g,℃

and 30 μg supernatant samples were loaded on the gel for electrophoresis and transfer.

Immunobloting of supercomplex was performed with anti-Complex I subunit

NDUFA9, anti-Complex III subunit Core2, and anti-complex IV subunit I (Invitrogen,

San Diego, CA).

SOD2 mutation and overexpression

Mouse Sod2 cDNA was cloned into pcDNA3.1 using BamHI/XhoI sites. Primer

sequences used for cloning were as follows: sod2 forward, 5'-

CGGGATCCGCCACCATGTTGTGTCGGGCGGCGTG-3'; sod2 reverse 5'-

CCGCTCGAGCGGCTACTTCTTGCAAGCTGTGT-3'. For SOD2 K68Q mutation,

the pcDNA-HA-SOD2 vector was amplified with the mutant primers (Forward: 5’-

CAACGCCACCGAGGAGCAGTACCACGAGGCTCTGG-3’; Reverse: 5’-

CCAGAGCCTCGTGGTACTGCTCCTCGGTGGCGTTG-3’). The PCR products

were digested with Dpn I, purified with agarose gel extract kit, self-ligated and were

transformed into DH5α. Colonies were randomly picked for plasmid extraction, and

K68Q mutation was verified through DNA sequencing. For wild type and mutant

SOD2 overexpression, stable SOD2 knockdown cells were achieved through shRNA

vector targeting 3’UTR of SOD2 mRNA (target sequence:

TCTATTCTTAGGCAACTATTT). After medium scale preparation, SOD2

knockdown cells were transfected with HA-tagged plasmids of both wild type and

mutant SOD2 using X-tremeGENE HP DNA transfection reagent (Roche, Penzberg,

Germany)

Supplementary Table. 1 Primer sequences used for qPCR analysis

Gene Primer sequence

Runx2Forward: 5’-TTCTCCAACCCACGAATGCAC-3’Reverse: 5’-CAGGTACGTGTGGTAGTGAGT-3’

OsterixForward: 5’-TCCCTACCCAGCGCCCCACCTCT-3’Reverse: 5’-CTGTGAATGGGCTTCTTCCTCAGC-3’

ALPForward: 5’-GTTGCCAAGCTGGGAAGAACAC-3’Reverse: 5’-CCCACCCCGCTATTCAAAC-3’

BSPForward: 5’-GAATCCACATGCCTATTGC-3’Reverse: 5’-AGAACCCACTGACCCATT-3’

OCNForward: 5’-GAACAGACTCCGGCGCTA-3’Reverse: 5’-AGGGAGGATCAAGTCCCG-3’

SOD1Forward: 5’-GGAAGCATGGCGATGAAAGC-3’Reverse: 5’-GCCTTCTGCTCGAAGTGGAT-3’

SOD2Forward: 5’-GCAGTGTGCGGCACCAGCAG-3’Reverse: 5’-TCCCTTGGCCAACGCCTCCT-3’

SIRT1Forward: 5’- AGAACCACCAAAGCGGAAA -3’Reverse: 5’- TCCCACAGGAGACAGAAACC -3’

SIRT2Forward: 5’-CAAGCCAACCATCTGCCACTA-3’Reverse: 5’-CCCGCCACTCGTTCCA-3’

SIRT3Forward: 5’- TGCTACTCATCTTGGGACCT-3’Reverse: 5’- CACCAGCCTTTCCACACC-3’

SIRT4Forward: 5’-GTCCCGTGCTGTGATCGA-3’Reverse: 5’-CGGGCGGTGAGGATGAAC-3’

SIRT5Forward: 5’-GCCTCCCCACAAAGCAAGA-3’Reverse: 5’-AACCCCACTCTCCGCACTAA-3’

SIRT6Forward: 5’-CCTGCCCCTTGCCACTAA-3’Reverse: 5’-GCACATCACCTCATCCACGTA-3’

SIRT7Forward: 5’-GCCAGGAGGAGGTGTGTGA-3’Reverse: 5’-GGCTCCGCTTCGCTTAGGT-3’

GAPDH Forward: 5’-GACTTCAACAGCAACTCCCAC-3’

Reverse: 5’-TCCACCACCCTGTTGCTGTA-3’

Supplementary Figures

Supplementary Figures.1

Supplementary Figure 1. Increased mitochondrial biogenesis in primary

osteoblasts differentiation. Calvaria-derived primary osteoblasts were isolated from

neonatal wild type mice and induced to differentiate for indicated period. (a) Seahorse

analysis of oxygen consumption rate. (b) Protein expression of mitochondrial

biogenesis regulator PGC-1α and mtTFA. (C) Protein expression of mitochondrial

complex subunits. Data were represented as mean±SEM. *P < 0.05, **P < 0.01 vs.

relative control.

Supplementary Figures.2

Supplementary Figure 2. Increased mitochondrial supercomplex formation

during osteoblasts differentiation. MC3T3-L1 cells were induced differentiation for

7 and 21 days, mitochondria were isolated and applied to BN-PAGE analysis,

supercomplex formation was presented by Western blot.

Supplementary Figures.3

Supplementary Figure 3. SiRT3 deacetylates K68 and promotes SOD2 activity in

osteoblasts. Stable knockdown of SOD2 was generated with shRNA targeting 3’UTR

in MC3T3-L1 cells, and overexpression of wild type SOD2 and SOD2 with K68

mutation were performed in the stable cells. (a) Protein expression of transient

transfection of SOD2 constructs. (b) ROS content was measured after 48 h

transfection. (c) Mitochondrial superoxide levels were visualized by fluorescence

staining after 48 h transfection. (d) ALP activity was measured from 48 h differentiated

cells after 48 h transfection. (e) Osteogenic markers were analyzed from 48 h

differentiated cells after 48 h transfection. Data were represented as mean±SEM. *P <

0.05, **P < 0.01 vs. relative control.

Supplementary Figure. 4

Supplementary Figure 4. SIRT3 knockdown impairs mitochondrial activity and

biogenesis in MC3T3-E1 cells. SIRT3 knockdown stable cells were generated with

specific shRNAs. (a) Sirt3 mRNA level. (b) SIRT3 protein expression. (c) T-AOC was

analyzed with whole cell homogenates. (d) Mitochondrial complex I and II activities

were analyzed with isolated mitochondria. (e) mtDNA copy number was analyzed by

qPCR. (f) Mitochondrial biogenesis was measured by Western blot. Data were

represented as mean±SEM. *P < 0.05, **P < 0.01 vs. relative control.

Supplementary Figures.5

Supplementary Figure 5. Impaired mitochondrial function and biogenesis in

Sirt3-/- primary osteoblasts. Calvaria-derived primary osteoblasts were isolated

from neonatal wild type and Sirt3-/- mice. (a) Seahorse analysis of oxygen

consumption rate. (b) Protein expression of PGC-1α, mtTFA and mitochondrial

complex subunits. (c) Primary osteoblasts were differentiated for 48 h, osteogenic

markers were analyzed by qPCR. Data were represented as mean±SEM. *P < 0.05,

**P < 0.01 vs. relative control.

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