Journal: Journal of Clinical Medicine

Article Title: Impact of Short-Term Hypoxia on Sirtuins as Regulatory Elements in HUVECs

doi: 10.3390/jcm9082604

Figure Lengend Snippet: Relative gene expression of sirtuins in human umbilical vein endothelial cells (HUVECs). Expression of SIRT1 ( A ), SIRT3 ( B ), and SIRT4 ( C ) in HUVECs. Controls under the normoxic conditions were compared with cells incubated under hypoxia for 10, 60, and 120 min. Shown are the median, upper, and lower quartile, and the 5% and 95% percentile. Each group had a sample size of n = 3. Significance: * = p < 0.05.

Article Snippet: Primary antibodies used were SIRT1 rabbit monoclonal (MerckMillipore, Merck KGaA, Darmstadt, Germany) (MW: ~130 kDa), SIRT3 rabbit polyclonal (MerckMillipore, Merck KGaA, Darmstadt, Germany) (MW: ~46 kDa); SIRT4 rabbit polyclonal (Santa Cruz Biotechnolgy, Inc., Dallas, USA) (MW: ~39 kDa); α-Tubulin rabbit polyclonal (Cell Signaling Technology, Inc., Danvers, USA) (MW: ~52 kDa).

Techniques: Expressing, Incubation

Journal: Journal of Clinical Medicine

Article Title: Impact of Short-Term Hypoxia on Sirtuins as Regulatory Elements in HUVECs

doi: 10.3390/jcm9082604

Figure Lengend Snippet: Western blot analysis and relative protein content of sirtuins in HUVECs. The relative protein content of SIRT1 ( A ), SIRT3 ( B ), and SIRT4 ( C ) in HUVECs. Controls under the normoxic conditions were compared with cells incubated under hypoxia for 10, 60, and 120 min. Shown are the median, upper, and lower quartile, and the 5% and 95% percentile. Each group had a sample size of n = 3. Significance: * p < 0.05 and ** p < 0.005. Representative Western blot analysis ( D ) of cells incubated under normoxia (21% O 2 ) and hypoxia (2% O 2 ) for 10, 60, and 120 min. α-Tubulin ~52kDa; SIRT1 ~110 kDa; SIRT3 ~44 kDa; SIRT4 ~39 kDa.

Article Snippet: Primary antibodies used were SIRT1 rabbit monoclonal (MerckMillipore, Merck KGaA, Darmstadt, Germany) (MW: ~130 kDa), SIRT3 rabbit polyclonal (MerckMillipore, Merck KGaA, Darmstadt, Germany) (MW: ~46 kDa); SIRT4 rabbit polyclonal (Santa Cruz Biotechnolgy, Inc., Dallas, USA) (MW: ~39 kDa); α-Tubulin rabbit polyclonal (Cell Signaling Technology, Inc., Danvers, USA) (MW: ~52 kDa).

Techniques: Western Blot, Incubation

Journal: Journal of Clinical Medicine

Article Title: Impact of Short-Term Hypoxia on Sirtuins as Regulatory Elements in HUVECs

doi: 10.3390/jcm9082604

Figure Lengend Snippet: Relative sirtuin enzyme capacity in the HUVECs. Relative maximum activity of SIRT1 ( A ) and SIRT3 ( B ) in the HUVECs. Normoxic controls were compared with cells incubated under hypoxia for 10, 60, and 120 min. Shown are the median, upper, and lower quartile, and the 5% and 95% percentile. Each group had a sample size of n = 3. Normoxic SIRT1 enzyme capacity averaged 1.8 U/10,000 cells in the normoxic HUVECs. The normoxic enzyme activity of SIRT3, averaged 9.5 U/µg protein.

Article Snippet: Primary antibodies used were SIRT1 rabbit monoclonal (MerckMillipore, Merck KGaA, Darmstadt, Germany) (MW: ~130 kDa), SIRT3 rabbit polyclonal (MerckMillipore, Merck KGaA, Darmstadt, Germany) (MW: ~46 kDa); SIRT4 rabbit polyclonal (Santa Cruz Biotechnolgy, Inc., Dallas, USA) (MW: ~39 kDa); α-Tubulin rabbit polyclonal (Cell Signaling Technology, Inc., Danvers, USA) (MW: ~52 kDa).

Techniques: Activity Assay, Incubation

Journal: The Journal of Clinical Investigation

Article Title: Sirt3 protects in vitro-fertilized mouse preimplantation embryos against oxidative stress-induced p53-mediated developmental arrest

doi: 10.1172/JCI42020

Figure Lengend Snippet: (A) Quantitative real-time RT-PCR analysis of sirtuin gene expression after H2O2 treatment (20 μM, 6 hours) in preimplantation embryos. Sirt3 mRNA levels were significantly increased in 4-cell embryos after exposure to H2O2. Data are derived from 3 independent experiments. Statistical assessments were performed by applying Mann-Whitney U test. *P < 0.05. (B and C) Western blotting analysis showing Sirt3 protein upregulation in 4-cell embryos (B) and NIH 3T3 cells (C) after H2O2 treatment (B, 20 and 100 μM, 18 hours; C, 20 μM, 24 hours) detected by Western blot. Blotting for α-tubulin served as an internal control.

Article Snippet: The separated proteins were transferred to a nylon membrane, which was then pretreated with 3% bovine serum albumin for blocking and incubated with primary antibodies as follows: rabbit anti-Sirt3 antibody (Abgent) for NIH 3T3 cell samples, rabbit anti-Sirt3 antibody from E. Verdin (UCSF, San Francisco, California, USA; ref. 26 ) for early embryo samples, and mouse anti-p53 antibody (BD Biosciences — Pharmingen).

Techniques: Quantitative RT-PCR, Expressing, Derivative Assay, MANN-WHITNEY, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Sirt3 protects in vitro-fertilized mouse preimplantation embryos against oxidative stress-induced p53-mediated developmental arrest

doi: 10.1172/JCI42020

Figure Lengend Snippet: Intracellular injection of mRNA coding Sirt3-EGFP (A–C), Sirt1-EGFP (D–F), Sirt2-EGFP (G–I), or EGFP alone (J) was performed at the pronuclear stage, and the EGFP signals (A, D, G, and J; green) were observed at the 2-cell stage. (B, E, and H) Mitochondria were stained by MitoTracker (red) just 15 minutes before observation. (C, F, and I) Colocalization of Sirt3-EGFP signals to MitoTracker staining confirmed mitochondrial localization of Sirt3. Scale bars: 20 μm.

Article Snippet: The separated proteins were transferred to a nylon membrane, which was then pretreated with 3% bovine serum albumin for blocking and incubated with primary antibodies as follows: rabbit anti-Sirt3 antibody (Abgent) for NIH 3T3 cell samples, rabbit anti-Sirt3 antibody from E. Verdin (UCSF, San Francisco, California, USA; ref. 26 ) for early embryo samples, and mouse anti-p53 antibody (BD Biosciences — Pharmingen).

Techniques: Injection, Staining

Journal: The Journal of Clinical Investigation

Article Title: Sirt3 protects in vitro-fertilized mouse preimplantation embryos against oxidative stress-induced p53-mediated developmental arrest

doi: 10.1172/JCI42020

Figure Lengend Snippet: (A and B) Injection of Sirt3 siRNA increased intracellular ROS levels, as estimated by CM-H2DCFDA fluorescence intensity. This increase was abolished by NAC (A) and stigmatellin (B), but was only partially decreased by apocynin (B). Embryos were injected with control or Sirt3 siRNA at the pronuclear stage and were cultured with or without NAC for 72 hours. To identify the major origin of increased intracellular ROS, embryos were treated with apocynin or stigmatellin for 30 minutes before CM-H2DCFDA staining. Quantitative data of fluorescence intensity, obtained using ImageJ, were standardized by dividing each value by the average value of the control group in each experiment. Data are derived from 3 (A) or 4 (B) independent experiments. Statistical assessments were performed by applying Games-Howell test. *P < 0.05; **P < 0.001. (C and D) Representative images of CM-H2DCFDA fluorescence in embryos analyzed in A and B, respectively. Scale bars: 100 μm.

Article Snippet: The separated proteins were transferred to a nylon membrane, which was then pretreated with 3% bovine serum albumin for blocking and incubated with primary antibodies as follows: rabbit anti-Sirt3 antibody (Abgent) for NIH 3T3 cell samples, rabbit anti-Sirt3 antibody from E. Verdin (UCSF, San Francisco, California, USA; ref. 26 ) for early embryo samples, and mouse anti-p53 antibody (BD Biosciences — Pharmingen).

Techniques: Injection, Fluorescence, Cell Culture, Staining, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: Sirt3 protects in vitro-fertilized mouse preimplantation embryos against oxidative stress-induced p53-mediated developmental arrest

doi: 10.1172/JCI42020

Figure Lengend Snippet: (A) Intracytoplasmic injection of both stealth Sirt3 siRNAs led to decreased blastocyst formation rate in preimplantation embryos. Data are derived from 4 independent experiments. (B) Sirt3 siRNA–induced decrease in blastocyst formation rate was suppressed by treatment with NAC. Data are derived from 6 independent experiments. Statistical assessments were performed by applying Ryan’s multiple-comparison test. *P < 0.05; **P < 0.001.

Article Snippet: The separated proteins were transferred to a nylon membrane, which was then pretreated with 3% bovine serum albumin for blocking and incubated with primary antibodies as follows: rabbit anti-Sirt3 antibody (Abgent) for NIH 3T3 cell samples, rabbit anti-Sirt3 antibody from E. Verdin (UCSF, San Francisco, California, USA; ref. 26 ) for early embryo samples, and mouse anti-p53 antibody (BD Biosciences — Pharmingen).

Techniques: Injection, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: Sirt3 protects in vitro-fertilized mouse preimplantation embryos against oxidative stress-induced p53-mediated developmental arrest

doi: 10.1172/JCI42020

Figure Lengend Snippet: (A) Intracytoplasmic injection of Sirt3 siRNA did not affect 2-cell and blastocyst formation rates in low-oxygen (5% O2) conditions. Data are derived from 4 independent experiments. (B) Effects of siRNA-mediated Sirt3 knockdown on intracellular ROS levels, as estimated by CM-H2DCFDA fluorescence intensity, in 20% and 5% O2 conditions. Sirt3 knockdown–induced ROS increases were canceled in 5% O2 conditions. Data are derived from 3 independent experiments. Statistical assessments were performed by applying Games-Howell test. *P < 0.05. (C) Representative images of CM-H2DCFDA fluorescence in embryos analyzed in B. Scale bar: 100 μm.

Article Snippet: The separated proteins were transferred to a nylon membrane, which was then pretreated with 3% bovine serum albumin for blocking and incubated with primary antibodies as follows: rabbit anti-Sirt3 antibody (Abgent) for NIH 3T3 cell samples, rabbit anti-Sirt3 antibody from E. Verdin (UCSF, San Francisco, California, USA; ref. 26 ) for early embryo samples, and mouse anti-p53 antibody (BD Biosciences — Pharmingen).

Techniques: Injection, Derivative Assay, Fluorescence

Journal: The Journal of Clinical Investigation

Article Title: Sirt3 protects in vitro-fertilized mouse preimplantation embryos against oxidative stress-induced p53-mediated developmental arrest

doi: 10.1172/JCI42020

Figure Lengend Snippet: Effect of maternal Sirt3 genotype on IVF rate

Article Snippet: The separated proteins were transferred to a nylon membrane, which was then pretreated with 3% bovine serum albumin for blocking and incubated with primary antibodies as follows: rabbit anti-Sirt3 antibody (Abgent) for NIH 3T3 cell samples, rabbit anti-Sirt3 antibody from E. Verdin (UCSF, San Francisco, California, USA; ref. 26 ) for early embryo samples, and mouse anti-p53 antibody (BD Biosciences — Pharmingen).

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Sirt3 protects in vitro-fertilized mouse preimplantation embryos against oxidative stress-induced p53-mediated developmental arrest

doi: 10.1172/JCI42020

Figure Lengend Snippet: Effect of maternal Sirt3 genotype on blastocyst formation rate after IVF

Article Snippet: The separated proteins were transferred to a nylon membrane, which was then pretreated with 3% bovine serum albumin for blocking and incubated with primary antibodies as follows: rabbit anti-Sirt3 antibody (Abgent) for NIH 3T3 cell samples, rabbit anti-Sirt3 antibody from E. Verdin (UCSF, San Francisco, California, USA; ref. 26 ) for early embryo samples, and mouse anti-p53 antibody (BD Biosciences — Pharmingen).

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: Sirt3 protects in vitro-fertilized mouse preimplantation embryos against oxidative stress-induced p53-mediated developmental arrest

doi: 10.1172/JCI42020

Figure Lengend Snippet: (A) After Sr2+ activation for 4.5 hours, eggs that formed 2 pronuclei were injected with control or Sirt3 siRNA, and the rates of 2-cell and blastocyst formation were evaluated. (B) Eggs collected from wild-type and Sirt3–/– female mice underwent parthenogenetic activation. Data are derived from 4 (A) or 2 (B) independent experiments. Statistical assessments were performed by applying Fisher’s exact test. **P < 0.001.

Article Snippet: The separated proteins were transferred to a nylon membrane, which was then pretreated with 3% bovine serum albumin for blocking and incubated with primary antibodies as follows: rabbit anti-Sirt3 antibody (Abgent) for NIH 3T3 cell samples, rabbit anti-Sirt3 antibody from E. Verdin (UCSF, San Francisco, California, USA; ref. 26 ) for early embryo samples, and mouse anti-p53 antibody (BD Biosciences — Pharmingen).

Techniques: Activation Assay, Injection, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: Sirt3 protects in vitro-fertilized mouse preimplantation embryos against oxidative stress-induced p53-mediated developmental arrest

doi: 10.1172/JCI42020

Figure Lengend Snippet: (A and B) Implantation rates (A) and full-term survival rates (B) of embryos injected with Sirt3 or control siRNA and transferred into pseudopregnant mice at the 2-cell or morula/blastocyst stage. Implantation sites and viability of fetuses were inspected by cesarean section 18 days after transfer. Implantation rate was estimated by the number of implantation sites; full-term survival rate was assessed by dividing the number of live pups by the number of implantation sites. Data are derived from 5 (2-cell embryo transfer) or 4 (morula/blastocyst transfer) independent experiments. Statistical assessments were performed by applying Ryan’s multiple-comparison test. *P < 0.005; **P < 0.001.

Article Snippet: The separated proteins were transferred to a nylon membrane, which was then pretreated with 3% bovine serum albumin for blocking and incubated with primary antibodies as follows: rabbit anti-Sirt3 antibody (Abgent) for NIH 3T3 cell samples, rabbit anti-Sirt3 antibody from E. Verdin (UCSF, San Francisco, California, USA; ref. 26 ) for early embryo samples, and mouse anti-p53 antibody (BD Biosciences — Pharmingen).

Techniques: Injection, Derivative Assay

Journal: The Journal of Clinical Investigation

Article Title: Sirt3 protects in vitro-fertilized mouse preimplantation embryos against oxidative stress-induced p53-mediated developmental arrest

doi: 10.1172/JCI42020

Figure Lengend Snippet: (A) p53 and p21 were upregulated in Sirt3–/– embryos as in H2O2-treated wild-type embryos. Nanog expression was decreased in Sirt3–/– embryos, and the decrease was enhanced by H2O2 stimulus. (B) Effects of Sirt3 knockdown and treatment with NAC on the expression of p53 and its downstream genes. In Sirt3 siRNA–injected embryos, p21 expression was upregulated, whereas Nanog expression was downregulated. These effects were blocked by NAC. (C) Western blotting analysis showing increased p53 protein levels in Sirt3-knockdown embryos at the morula stage. Signals for acetylated histone H3 (Ac-H3) served as an internal control. (D) Effects of p53 knockdown on Sirt3 siRNA–induced changes in the expression of genes downstream of p53. Sirt3 siRNA–induced p21 upregulation and Nanog downregulation were blocked by siRNA-mediated p53 knockdown. Ppia expression served as an internal control in A, B, and D. (E) Effects of p53 knockdown on preimplantation developmental arrest in Sirt3-knockdown embryos. The rate of blastocyst formation was significantly improved by coinjection with p53 siRNA. Data are derived from 4 independent experiments. Statistical assessments were performed by applying Ryan’s multiple-comparison test. *P < 0.05; **P < 0.001.

Article Snippet: The separated proteins were transferred to a nylon membrane, which was then pretreated with 3% bovine serum albumin for blocking and incubated with primary antibodies as follows: rabbit anti-Sirt3 antibody (Abgent) for NIH 3T3 cell samples, rabbit anti-Sirt3 antibody from E. Verdin (UCSF, San Francisco, California, USA; ref. 26 ) for early embryo samples, and mouse anti-p53 antibody (BD Biosciences — Pharmingen).

Techniques: Expressing, Injection, Western Blot, Derivative Assay

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