Journal: PLoS ONE

Article Title: Changes in Muscle Cell Metabolism and Mechanotransduction Are Associated with Myopathic Phenotype in a Mouse Model of Collagen VI Deficiency

doi: 10.1371/journal.pone.0056716

Figure Lengend Snippet: Histograms and representative immunoblot images of FASN (panel A), PPARγ, α and β (panel C), PGC1-α (panel D), SIRT1 and SIRT3 (panel E) in gastrocnemius, tibialis anterior and diaphragm from Col6a1 −/− and wild-type mice (n = 4; mean ± S.D.; Student’s T-test). Panel B: electron microscopy image showing lipid accumulation in the diaphragm of Col6a1 −/− mice. Black arrows show lipid droplets.

Article Snippet: Blots were incubated with rabbit or goat polyclonal primary antibodies (Cell Signaling Technology and Santa Cruz Biotechnology) as follows: anti-TN-C (1∶500), anti-ROCK1 (1∶500), anti PI3K (1∶1000), anti FAK (1∶1000), anti-PPARγ (1∶1000), anti-PPARα (1∶1000), anti-PPARβ (1∶1000), anti-Sirt1 (1∶1000), anti-Sirt3 (1∶1000), anti-FASN (1∶1000), anti-IDH1 (1∶1000), anti-IDH2 (1∶1000) and anti β-tubulin (1∶1000).

Techniques: Western Blot, Electron Microscopy

Journal: Molecular Neurodegeneration

Article Title: Alpha-synuclein-induced mitochondrial dysfunction is mediated via a sirtuin 3-dependent pathway

doi: 10.1186/s13024-019-0349-x

Figure Lengend Snippet: Antibodies used for western blot and immunocyhistochemistry

Article Snippet: SIRT3 (mouse) , Santa Cruz (sc-135,796) , 1:1000 (WB).

Techniques: Western Blot, Transduction

Journal: Molecular Neurodegeneration

Article Title: Alpha-synuclein-induced mitochondrial dysfunction is mediated via a sirtuin 3-dependent pathway

doi: 10.1186/s13024-019-0349-x

Figure Lengend Snippet: αSyn is found in mitochondria-enriched fraction of H4 SL1&SL2 cells and induces a decrease in SIRT3 expression. a Representative cropped western blots showing αsyn and SIRT3 in cytosolic and mitochondrial fractions at different time points (0 – 72 h) ( b ) αsyn oligomers are apparent after 24 h by luciferase assay (RLU: relative luminescence units), n = 5. Quantification of SIRT3 protein level in mitochondria demonstrates significant decrease in SIRT3 at 48 h and 72 h. c Whole cells lysates from H4 SL1&SL2 cells demonstrate decreased SIRT3 expression after transfection with SIRT3 siRNA n = 6 ( d ) Luciferase activity from αsyn oligonmerization is significantly increased in cells transfected with SIRT3 siRNA (siSIRT3) compared to control siRNA (siCtrl). Error bars represent the mean ± SD. * p < 0.05, ** p < 0.01. Note: In ( a ) αsyn and SIRT3 bands are from different experiments run on different gels. COXIV, GAPDH, and SIRT3 are all from same samples and immunoblot. Loading controls for αsyn blot are not shown. In panel ( c ) αsyn, SIRT3, and GAPDH are detected on same immunoblot

Article Snippet: SIRT3 (mouse) , Santa Cruz (sc-135,796) , 1:1000 (WB).

Techniques: Expressing, Western Blot, Luciferase, Transfection, Activity Assay, Control

Journal: Molecular Neurodegeneration

Article Title: Alpha-synuclein-induced mitochondrial dysfunction is mediated via a sirtuin 3-dependent pathway

doi: 10.1186/s13024-019-0349-x

Figure Lengend Snippet: AICAR activates AMPK-CREB signaling pathway and increases SIRT3 activity and reduces αsyn oligomers. a Representative cropped western blots showing AMPKα, p-AMPKα (Thr 172), CREB, and p-CREB (Ser 133) in H4 SL1&SL2 cells with/without 2 mM AICAR. b SIRT3 expression increases with AICAR-treatment, n = 3. c Luciferase assay ( n = 5) demonstrates AICAR significantly decreases αsyn oligomers and knockdown of SIRT3 (siSIRT3) prevents reduction of αsyn oligomers by AICAR. d Native-page shows AICAR significantly decreases αsyn oligomers. e Representative cropped western blot showing increased Ac-SOD2 (acetyl K68) with no change in total SOD2 in whole cells lysates. AICAR restored acetylated SOD2 levels, n = 3. Error bars represent the mean ± SD, ( n = 3–5). * p < 0.05, ** p < 0.01. In panel ( a ) the same samples were run on different gels and probed separately for AMPKα, p-AMPKα, and GAPDH, and CREB, p-CREB, and GAPDH respectively. In panels ( b ) and ( e ) separate blots were probed for SIRT3 and GAPDH, and COXIV, or SOD2, Ac-SOD2, and GAPDH

Article Snippet: SIRT3 (mouse) , Santa Cruz (sc-135,796) , 1:1000 (WB).

Techniques: Activity Assay, Western Blot, Expressing, Luciferase, Knockdown, Clear Native PAGE

Journal: Molecular Neurodegeneration

Article Title: Alpha-synuclein-induced mitochondrial dysfunction is mediated via a sirtuin 3-dependent pathway

doi: 10.1186/s13024-019-0349-x

Figure Lengend Snippet: Activation of SIRT3 by AICAR attenuates ROS production. a Fluorescence microscopy images of MitoSox-Red and Mitotracker-Green staining in fixed H4 SL1&SL2 cells. MitoSox-Red fluorescence increases when αsyn is overexpressed and AICAR treatment attenuates mtROS. Representative images from 3 experiments. MitoTracker-Green (mitochondria; green); MitoSox-Red (mitochondria; red); merged images (yellow). Scale bar = 10 μm. b Relative intensity of MitoTracker-Green signal between conditions. Quantification of MitoSOX-Red signal intensities ( n = 5), and mean intensity of MitoSOX-Red normalized to MitoTracker-Green intensity ( c ) Representative cropped western blot of HO-1 and GAPDH in whole cells lysates from H4 SL1&SL2 cells. HO-1 level increases at 72 h and is reduced after AICAR-treatment, n = 5. Error bars represent the mean ± SD. * p < 0.05, ** p < 0.01

Article Snippet: SIRT3 (mouse) , Santa Cruz (sc-135,796) , 1:1000 (WB).

Techniques: Activation Assay, Fluorescence, Microscopy, Staining, Western Blot

Journal: Molecular Neurodegeneration

Article Title: Alpha-synuclein-induced mitochondrial dysfunction is mediated via a sirtuin 3-dependent pathway

doi: 10.1186/s13024-019-0349-x

Figure Lengend Snippet: αSyn expression decreases SIRT3 and alters mitochondrial dynamics in vivo. a αSyn oligomers were quantified via luciferase assay in rat brain homogenates at 4 weeks after stereotaxic injection of AAV8-SL1&SL2 in SN, n = 5. b Representative cropped western blots showing αsyn, SIRT3, DRP1 in cytosol and mitochondria from SN of rats 4 weeks after stereotaxic injection of AAV8-SL1 and AAV8-SL2. (C). c Quantification of αsyn, SIRT3, DRP1, protein levels in cytosol and mitochondria from two separate blots for each of 4–5 rats. All bands were normalized to respective loading controls GAPDH and COXIV. d Representative cropped western blot of αsyn, DRP1, and p-DRP1 (Ser 616) in SN lysate of AAV8-SL1&SL2 injected rat. αSyn expression leads to increased p-DRP1 protein levels in ipsilateral (I) injected SN compared to contralateral (C) uninjected SN, n = 5 rats total. In panel ( a ) the same samples were run on one blot that was cropped prior to immunoblotting for αsyn, SIRT3, COXIV, DRP1, and GAPDH. Error bars represent the mean ± SD, ( n = 4–5 rats). * p < 0.05, ** p < 0.01

Article Snippet: SIRT3 (mouse) , Santa Cruz (sc-135,796) , 1:1000 (WB).

Techniques: Expressing, In Vivo, Luciferase, Injection, Western Blot

Journal: Molecular Neurodegeneration

Article Title: Alpha-synuclein-induced mitochondrial dysfunction is mediated via a sirtuin 3-dependent pathway

doi: 10.1186/s13024-019-0349-x

Figure Lengend Snippet: SIRT3 is decreased in human post-mortem brain of neuropathologically confirmed Lewy body disease individuals. a Representative cropped western blot from n = 3 showing decreased SIRT3 in total brain lysates from ten LBD brains compared to ten age-matched healthy controls. b Quantification of αsyn, SIRT3 protein levels from n = 3 western blots. c , d Representative western blot of cytosolic fraction. DRP1 and αsyn were quantified using GAPDH as a loading control. e , f Representative western blot of mitochondrial fraction. DRP1 and αsyn were quantified using COXIV as a loading control. Error bars represent the mean ± SD. ** p < 0.01

Article Snippet: SIRT3 (mouse) , Santa Cruz (sc-135,796) , 1:1000 (WB).

Techniques: Western Blot, Control

Journal: Molecular Neurodegeneration

Article Title: Alpha-synuclein-induced mitochondrial dysfunction is mediated via a sirtuin 3-dependent pathway

doi: 10.1186/s13024-019-0349-x

Figure Lengend Snippet: Cartoon representing of αsyn-induced effect on SIRT3 and mitochondrial dysfunction. Consequences of decreased SIRT3 include decreased AMPK-CREB signaling, impairment in mitochondrial bioenergetics and dynamics, and increased acetylation of SIRT3 substrates such as SOD2 all of which contribute to increased ROS production and neurodegeneration. Question mark indicates pathway not supported by data in this manuscript

Article Snippet: SIRT3 (mouse) , Santa Cruz (sc-135,796) , 1:1000 (WB).

Techniques:

Journal: Cell reports

Article Title: Intense Light-Mediated Circadian Cardioprotection via Transcriptional Reprogramming of the Endothelium

doi: 10.1016/j.celrep.2019.07.020

Figure Lengend Snippet: HMEC-1 or stable lentiviral-mediated PER2KD and Scr control HMEC-1 were synchronized and exposed to 24 h of normoxia (Nx) or 1% hypoxia (Hx). In a subset of experiments, synchronized stable lentiviral-mediated HIF1AKD and Scr HMEC-1 were exposed to Nx or Hx. (A and B) Affinity purification-mass spectrometry-based proteomics screen for PER2 protein interactions in normoxic and hypoxic HMEC-1. (A) Number of PER2 proteins regulated. (B) Pathways analysis using Ingenuity. (C and D) Coimmunoprecipitation for PER2 in hypoxic or normoxic HMEC-1 against isocitrate dehydrogenase (IDH) 2, succinyl coenzyme A (CoA) ligase (SUCLG) 1, and aconitase (ACO) 2 (C), and vice versa (D). One representative blot of three is displayed. (E) Subcellular compartment analysis of PER2 during normoxia or hypoxia (C, cytoplasm; N, nucleus; M, mitochondria; compartment-specific loading controls: tubulin alpha 1a (TUBA1A) for cytoplasm, TATA-box binding protein (TBP) for nucleus, and voltage-dependent anion channel 1 (VDAC1) for mitochondria). (F) Translocation of PER2 into the mitochondria during hypoxia (scale bar, 20 μm). (G–I) TCA cycle enzyme activities of IDH (G), SUCLG (H), and ACO (I) from stable lentiviral-mediated PER2KD and Scr control HMEC-1 during hypoxia (mean ± SD; n = 3; Student’s t test). (J) Carbon dioxide evolution rate (CDER), as a surrogate for TCA cycle function, in PER2KD or Scr HMEC-1 measured by a mitochondrial stress test using a Seahorse XF24 FluxPak assay (mean ± SD; n = 5; Student’s t test). (K–M) SIRT3 transcript (K and L) or protein (M) levels from stable lentiviral-mediated PER2KD and Scr (K and M, upper panel) or stable lentiviral-mediated HIF1AKD and Scr (L and M, lower panel) control HMEC-1 (mean ± SD; n = 3; ANOVA with Tukey’s multiple comparison test). See also – .

Article Snippet: The primary antibodies used were rabbit polyclonal PER2 (Novus Biologicals, NB100-125, Littleton CO, or Abcam, ab64460, Cambridge, MA), mouse monoclonal actin (Ab-1) (JLA20, Calbiochem, Diego, CA,), rabbit polyclonal IDH2 (Novus Biologicals, NBP2-22166, Littleton CO), rabbit polyclonal SUCLG1 (Novus Biologicals, NBP1089489, Littleton CO), rabbit polyclonal ACO2 (Novus Biologicals, H00000050-D01P, Littleton CO), rabbit polyclonal SIRT3 (Abcam, ab86671, Cambridge, MA), anti-alpha Tubulin antibody (Abcam, ab7291, Cambridge, MA), Anti-VDAC1 / Porin antibody (Abcam, ab15895, Cambridge, MA), Anti-TATA binding protein (TBP) antibody (Abcam, ab51841, Cambridge, MA), mouse monoclonal β-ACTIN (Cell Signaling Technologies, 8H10D10, Danvers, MA), and mouse monoclonal anti-DDK (FLAG) (OriGene Technologies, TA50011-100, Rockville, MD).

Techniques: Control, Affinity Purification, Mass Spectrometry, Binding Assay, Translocation Assay, Comparison

Journal: Cell reports

Article Title: Intense Light-Mediated Circadian Cardioprotection via Transcriptional Reprogramming of the Endothelium

doi: 10.1016/j.celrep.2019.07.020

Figure Lengend Snippet: KEY RESOURCE TABLE

Article Snippet: The primary antibodies used were rabbit polyclonal PER2 (Novus Biologicals, NB100-125, Littleton CO, or Abcam, ab64460, Cambridge, MA), mouse monoclonal actin (Ab-1) (JLA20, Calbiochem, Diego, CA,), rabbit polyclonal IDH2 (Novus Biologicals, NBP2-22166, Littleton CO), rabbit polyclonal SUCLG1 (Novus Biologicals, NBP1089489, Littleton CO), rabbit polyclonal ACO2 (Novus Biologicals, H00000050-D01P, Littleton CO), rabbit polyclonal SIRT3 (Abcam, ab86671, Cambridge, MA), anti-alpha Tubulin antibody (Abcam, ab7291, Cambridge, MA), Anti-VDAC1 / Porin antibody (Abcam, ab15895, Cambridge, MA), Anti-TATA binding protein (TBP) antibody (Abcam, ab51841, Cambridge, MA), mouse monoclonal β-ACTIN (Cell Signaling Technologies, 8H10D10, Danvers, MA), and mouse monoclonal anti-DDK (FLAG) (OriGene Technologies, TA50011-100, Rockville, MD).

Techniques: Binding Assay, Virus, Variant Assay, Recombinant, Protein Extraction, Extraction, Isolation, Cell Culture, Membrane, Transfection, Enzyme-linked Immunosorbent Assay, Reporter Assay, Activity Assay, Colorimetric Assay, Transcription Factor Assay, Bicinchoninic Acid Protein Assay, Chromatin Immunoprecipitation, Qubit Protein Assay, SYBR Green Assay, LDH Cytotoxicity Assay, Microarray, Luciferase, Generated, shRNA, Sequencing, Control, Software

Journal: bioRxiv

Article Title: Endogenous SIRT3 activity is dispensable for normal hearing recovery after noise exposure in young adult mice

doi: 10.1101/2020.02.05.935882

Figure Lengend Snippet: a. Cochlear whole mount preparation from a wild-type adult mouse, immunolabeled with antibodies specific to MYO7 (green) and SIRT3 (red), and co-stained with DAPI (blue) to reveal nuclei. The vertical yellow lines show the positions from which the side views are taken. (a’) shows anti-SIRT3 immunoreactivity only. b. Cochlear whole mount preparation from a Sirt3-KO adult mouse, identically labeled and imaged. This control was used to set the background to reveal SIRT3-specific staining.

Article Snippet: The following primary antibodies were used: mouse IgG1 anti-SIRT3 antibody (1:200; Novus Biologicals; RRID:AB_2818991); goat anti-Oncomodulin antibody (OCM; 1:1000; Santa Cruz; RRID:AB_2267583), rabbit anti-Myosin7a (MYO7; 1:200; Proteus; RRID:AB_10013626) mouse anti-CTBP2 (aka C-Terminal Binding Protein 2; 1:200; BD Transduction Laboratories; RRID:AB_399431), and mouse anti-GRIA2 (aka GluR2/GluA2; 1:2000; Millipore; RRID:AB_2113875).

Techniques: Immunolabeling, Staining, Labeling, Control

Journal: bioRxiv

Article Title: Endogenous SIRT3 activity is dispensable for normal hearing recovery after noise exposure in young adult mice

doi: 10.1101/2020.02.05.935882

Figure Lengend Snippet: a. The time course of experiments is shown. Homozygous Sirt3-KO mice and wild-type littermates are tested for ABR and DPOAE thresholds prior to noise exposure (pre-test). At P60, they are exposed to an 8-16 octave band noise at 105 dB for 30 minutes (noise), which is sufficient to drive temporary threshold shifts in this strain [ , ]. Their hearing thresholds are assessed again at 1 day post noise (1 DPN) and 14 days post noise (14 DPN). ABR and DPOAE are assessed at 8, 12, 16, 24, and 32 kHz for all panels. b. Mean ABR thresholds prior to noise exposure for homozygous Sirt3-KO mice (n=14, pink) and wild-type littermates (n=13, black). Mice were approximately P50 when tested. Homozygous Sirt3-KO mice had significantly worse overall hearing (p=0.004 for genotype, two way ANOVA, n=27 mice total); however, no single frequency was significantly worse (p=0.053, 0.39, 0.41, 0.53, 0.059 for the five frequencies respectively, two-tailed pairwise t-tests at each frequency with Bonferroni correction, n=27 mice total). c. Mean ABR thresholds at 1 DPN for homozygous Sirt3-KO mice (n=14, pink) and wild-type littermates (n=13, black). No significant difference was seen between genotypes (p=0.35 for genotype, two-way ANOVA, n=27 mice total). d. Mean ABR thresholds at 14 DPN for homozygous Sirt3-KO mice (n=14, pink) and wild-type littermates (n=13, black). No significant difference was seen between genotypes (p=0.85 for genotype, two way ANOVA, n=27 mice total). Overall ANOVA for all three time points showed no significant difference between genotypes (p=0.33, multi-way ANOVA, n=27 mice total). e. Mean DPOAE thresholds prior to noise exposure for homozygous Sirt3-KO mice (n=14, pink) and wild-type littermates (n=13, black). No significant difference was seen between genotypes (p=0.43 for genotype, two-way ANOVA, n=27 mice total). f. Mean DPOAE thresholds at 1 DPN for homozygous Sirt3-KO mice (n=14, pink) and wild-type littermates (n=13, black). No significant difference was seen between genotypes (p=0.50 for genotype, two-way ANOVA, n=27 mice total). g. Mean DPOAE thresholds at 14 DPN for homozygous Sirt3-KO mice (n=14, pink) and wild-type littermates (n=13, black). No significant difference was seen between genotypes (p=0.25 for genotype, two-way ANOVA, n=27 mice total). Error bars: s.e.m.

Article Snippet: The following primary antibodies were used: mouse IgG1 anti-SIRT3 antibody (1:200; Novus Biologicals; RRID:AB_2818991); goat anti-Oncomodulin antibody (OCM; 1:1000; Santa Cruz; RRID:AB_2267583), rabbit anti-Myosin7a (MYO7; 1:200; Proteus; RRID:AB_10013626) mouse anti-CTBP2 (aka C-Terminal Binding Protein 2; 1:200; BD Transduction Laboratories; RRID:AB_399431), and mouse anti-GRIA2 (aka GluR2/GluA2; 1:2000; Millipore; RRID:AB_2113875).

Techniques: Two Tailed Test

Journal: bioRxiv

Article Title: Endogenous SIRT3 activity is dispensable for normal hearing recovery after noise exposure in young adult mice

doi: 10.1101/2020.02.05.935882

Figure Lengend Snippet: a. Peak 1 amplitudes in ABR measurements for 32 kHz stimuli prior to noise exposure for homozygous Sirt3-KO mice (n=14, thin red) and wild-type littermates (n=13, thin black). Peak 1 amplitudes (microvolts) are plotted on the y axis for different amplitudes of stimulus (dB SPL) plotted on the x-axis. Heavier red and black lines represent respective GEE models. The area under the curve, representative of the progressive neuronal recruitment, is significantly reduced for homozygous Sirt3-KO mice (p=0.046, parametric bootstrap method, n=27 mice). b. Same analysis as in (a), but for peak 1 amplitude values obtained from the same mice 14 DPN. No difference is seen between genotypes (p=0.87, parametric bootstrap method, n=27 mice). c. Same analysis as in (a, b), however, in this case amplitudes obtained from homozygous Sirt3-KO mice are compared before and after noise exposure. No differences are seen (p=0.914, parametric bootstrap method, n=14). c. Same analysis as in (a, b), however, in this case amplitudes obtained from wild-type littermates are compared before and after noise exposure. A significant reduction in amplitude is evident after noise exposure (cf. red to black, p=0.002, parametric bootstrap method, n=13). e. Mean peak 1 latencies for 32 kHz stimuli prior to noise exposure for homozygous Sirt3-KO mice (n=14, pink) and wild-type littermates (n=13, black). No differences are seen between genotypes. f. Mean peak 1 latencies for 32 kHz stimuli at 14 DPN for homozygous Sirt3-KO mice (n=14, pink) and wild-type littermates (n=13, black). No differences are seen between genotypes. Error bars: s.e.m.

Article Snippet: The following primary antibodies were used: mouse IgG1 anti-SIRT3 antibody (1:200; Novus Biologicals; RRID:AB_2818991); goat anti-Oncomodulin antibody (OCM; 1:1000; Santa Cruz; RRID:AB_2267583), rabbit anti-Myosin7a (MYO7; 1:200; Proteus; RRID:AB_10013626) mouse anti-CTBP2 (aka C-Terminal Binding Protein 2; 1:200; BD Transduction Laboratories; RRID:AB_399431), and mouse anti-GRIA2 (aka GluR2/GluA2; 1:2000; Millipore; RRID:AB_2113875).

Techniques:

Journal: bioRxiv

Article Title: Endogenous SIRT3 activity is dispensable for normal hearing recovery after noise exposure in young adult mice

doi: 10.1101/2020.02.05.935882

Figure Lengend Snippet: a. Representative cochlear preparation from a wild-type littermate without noise exposure, with IHCs revealed with anti MYO7 antibodies (white) and OHCs revealed with anti-OCM antibodies (also white). Scale bar: 200 microns. b. Cochleogram results from 4 mapped cochlear preparations from 4 wild-type littermates without noise exposure, where OHC loss was quantified in 100 micron segments. The distance from the apex for each segment is plotted on the x-axis, and the percent OHC loss for the segment is plotted on the y-axis. Dots with the same color (purple, blue, green, or red) are from the same cochlear preparation. c. Representative cochlear preparation from a wild-type littermate at 14 DPN, with the same staining as in (a). d. Cochleogram results, similar to (b), from 3 mapped cochlear preparations from 3 wild-type littermates at 14 DPN. Results are similar to (b). e. Representative cochlear preparation from a homozygous Sirt3-KO mouse without noise exposure, with the same staining as in (a). f. Cochleogram results, similar to (b), from 4 mapped cochlear preparations from 4 homozygous Sirt3-KO mice without noise exposure. Results are similar to (b). g. Representative cochlear preparation from a homozygous Sirt3-KO mouse at 14 DPN, with the same staining as in (a). h. Cochleogram results, similar to (b), from 4 mapped cochlear preparations from 4 homozygous Sirt3-KO mice at 14 DPN. Results are similar to (f) and (b).

Article Snippet: The following primary antibodies were used: mouse IgG1 anti-SIRT3 antibody (1:200; Novus Biologicals; RRID:AB_2818991); goat anti-Oncomodulin antibody (OCM; 1:1000; Santa Cruz; RRID:AB_2267583), rabbit anti-Myosin7a (MYO7; 1:200; Proteus; RRID:AB_10013626) mouse anti-CTBP2 (aka C-Terminal Binding Protein 2; 1:200; BD Transduction Laboratories; RRID:AB_399431), and mouse anti-GRIA2 (aka GluR2/GluA2; 1:2000; Millipore; RRID:AB_2113875).

Techniques: Staining

Journal: bioRxiv

Article Title: Endogenous SIRT3 activity is dispensable for normal hearing recovery after noise exposure in young adult mice

doi: 10.1101/2020.02.05.935882

Figure Lengend Snippet: a. Representative image of wild-type 12 kHz IHCs with no noise exposure, stained with anti-MYO7 (blue), anti-CTBP2 (red) to reveal pre-synaptic structures, and anti-GRIA2 (green) to reveal post-synaptic structures. Mean synaptic number per IHC (± s.e.m.), visually counted from 3 biological replicates with 8 IHCs each, is noted in the upper right hand corner. Size bar: 5 microns. b. Representative image of wild-type 24 kHz IHCs with no noise exposure, similarly stained to (a). 3 replicates, 6-7 IHCs each. c. Representative image of wild-type 12 kHz IHCs at 14 DPN, similarly stained to (a). 4 replicates, 7-11 IHCs each. d. Representative image of wild-type 24 kHz IHCs at 14 DPN, similarly stained to (a). 3 replicates, 7-8 IHCs each. e – h. Three dimensional Amira rendering of confocal stacks shown in (a – d), respectively, with MYO7 rendered in white, CTBP2 in red, and GRIA2 in green. Hair cells are rotated to display from similar orientations. i. Representative image of homozygous Sirt3-KO 12 kHz IHCs with no noise exposure, similarly stained to (a). 3 replicates, 6-8 IHCs each. j. Representative image of homozygous Sirt3-KO 24 kHz IHCs with no noise exposure, similarly stained to (a). 3 replicates, 4-7 IHCs each. k. Representative image of homozygous Sirt3-KO 12 kHz IHCs at 14 DPN, similarly stained to (a). The angle of the tissue in the imaging foreshortens the IHC. 5 replicates, 6-8 IHCs each. l. Representative image of homozygous Sirt3-KO 24 kHz IHCs at 14 DPN, similarly stained to (a). IHCs appear different because of the angle of the tissue. 3 replicates, 6-8 IHCs each. m - p. Three-dimensional Amira rendering of confocal stacks shown in (i – l), respectively, with MYO7 rendered in white, CTBP2 in red, and GRIA2 in green. Hair cells are rotated to display from similar orientations. The apical truncation shown in (p) is an artifact of the confocal imaging.

Article Snippet: The following primary antibodies were used: mouse IgG1 anti-SIRT3 antibody (1:200; Novus Biologicals; RRID:AB_2818991); goat anti-Oncomodulin antibody (OCM; 1:1000; Santa Cruz; RRID:AB_2267583), rabbit anti-Myosin7a (MYO7; 1:200; Proteus; RRID:AB_10013626) mouse anti-CTBP2 (aka C-Terminal Binding Protein 2; 1:200; BD Transduction Laboratories; RRID:AB_399431), and mouse anti-GRIA2 (aka GluR2/GluA2; 1:2000; Millipore; RRID:AB_2113875).

Techniques: Staining, Imaging