Journal: bioRxiv

Article Title: SIRT3 deficiency decreases oxidative-metabolism capacity but increases lifespan under caloric restriction

doi: 10.1101/2022.05.09.491205

Figure Lengend Snippet: A) Immunoblot of pan-acetylation, normalized to VDAC, for liver mitochondrial enrichment from 25-month-old treatment groups. Data were analyzed by two-way ANOVA followed by multiple comparisons test. p value reported for each comparison is corrected by Tukey’s test. Results are plotted as mean ± SEM. *: p≤0.05. B) Percentage of significantly changed acetyl-lysine residues that show increased stoichiometry due to Sirt3 -/- , calculated by (the number of acetyl-lysine sites showing increased stoichiometry) / (the number of significantly changed acetyl-lysine sites, p<0.05) x100%. C) Heat map of significantly changed lysine sites (p<0.1) that are response to loss of SIRT3. Plotted sites are significantly changed (p<0.1) in either Sirt3 -/- CD vs. WTCD or Sirt3 -/- CR vs. WTCR comparison. Values are colored based on relative acetylation stoichiometry, normalized to the median value of each sites in all four groups, scaling ranging from -0.8 to 0.8 (x100%). D) Functional cluster analysis of KEGG pathways (DAVID 6.8). Significantly enriched (-log10(p value) >1.5) pathways are indicated, with Sirt3 -/- CD vs. WTCD in orange and Sirt3 -/- CR vs. WTCR in blue. E) Acetylation sites in FAO and BACC metabolism, TCA cycle, and ETC that displayed larger than 5% stoichiometry (p<0.1) for Sirt3 -/- CD vs. WTCD (orange colored) and Sirt3 -/- CR vs. WTCR comparison (blue colored).

Article Snippet: Western blot primary antibodies used include: SIRT3 (#5490, CST, 1:1000), Acetylated-Lysine (#9681, CST, 1:1000), VDAC (75-204, NeuroMab, 1:1250).

Techniques: Western Blot, Comparison, Functional Assay

Journal: Nature Communications

Article Title: Autophagy regulates fatty acid availability for oxidative phosphorylation through mitochondria-endoplasmic reticulum contact sites

doi: 10.1038/s41467-020-17882-2

Figure Lengend Snippet: a MOLM14 cells treated with metformin (Met, 10 mM) for 24 h were fixed and processed for electron microscopy analysis. Representative electron microscopy pictures from two independent experiments are shown. Arrows indicate MERCs. Scale bar: 1 µm. b Histograms represent the % of mitochondria that are in contact with endoplasmic reticulum per cell treated or not with Met from pictures displayed in a . Data are means ± s.e.m with each dot corresponding to one cell. c Protein components of subcellular fractions (left panel) from two independent experiments prepared from MOLM14 cells revealed by immunoblot analysis (right panel). TL: total lysate, Cy: cytosol, Mt: pure mitochondrial fraction, MERCs: mitochondria–ER contact site fraction. d Representative orthogonal confocal projections of Z sections of PLA (red signal) between VDAC1 and IP3R1 from MOLM14 cells treated or not with Met or antimycin A (AA) for 48 h (at least n = 3). Scale bar: 10 µm e Quantitative analysis of PLA signal between VDAC1 and IP3R1 from MOLM14 cells treated or not with Met or AA for 6 h, 24 h or 48 h ( n = 4, one-sample t -test). Data are means ± s.e.m.

Article Snippet: Mouse antibodies from Santa Cruz Biotechnology against IP3R1 (sc-271197) and E2F1 (KH95), from Millipore against actin, rabbit antibody from Abcam against VDAC1 (ab15865), from PROGEN against ADRP (610102), from R&D System against FABP4 (AF3150), from Atlas Antibodies against ACLY (HPA022434), from Novus Biologicals against SREPB1 (NB600-582), from Invitrogen against SREBP2 (PA1-338), and from GeneTex against TOMM20 (GTX133756) were also used.

Techniques: Electron Microscopy, Western Blot

Journal: Nature Communications

Article Title: Autophagy regulates fatty acid availability for oxidative phosphorylation through mitochondria-endoplasmic reticulum contact sites

doi: 10.1038/s41467-020-17882-2

Figure Lengend Snippet: a MOLM14 cells transfected with siRNA control (Ctrl) or mitofusin2 (Mtfn2) were processed for electron microscopy analysis. Electron microscopy pictures from one experiment are shown. Scale bar: 1 µm. b Histograms represent the % of mitochondria in contact with ER. Each dot corresponding to one cell. c Images of proximity ligation assay between VDAC1 and IP3R1 from MOLM14 transfected with Ctrl or Mtfn2 siRNAs ( n = 3). Scale bar: 10 µm. Numbers represent the % of dots in cells transfected with siRNA Mtfn2 compared with cells transfected with siRNA Ctrl. d , e MOLM14 cells were transduced with Ctrl or VDAC1 shRNAs ( n = 4) or transfected with Ctrl or Mtfn2 siRNAs ( n = 6), and stained for LC3B and DAPI. Confocal sections (at least four independent experiments) are shown. Scale bar: 10 µm ( d ). Histograms represent the number of LC3B puncta per cell ( e ) (unpaired t -test). f , g MOLM14 cells transduced with Ctrl or VDAC1 shRNAs ( n = 3) or transfected with Ctrl or Mtfn2 siRNAs ( n = 6) were stained for Bodipy and DAPI. Scale bar: 10 µm, ( f ). Histograms show the number or the area of Bodipy dots per cell ( g ) (unpaired t -test). h Measurement of basal oxygen consumption rate (OCR) in MOLM14 cells transduced with Ctrl or VDAC1 shRNAs ( n = 6) or transfected with Ctrl or Mtfn2 siRNAs ( n = 6, unpaired t -test). i , j MOLM14 cells transfected with Ctrl or Beclin1 (Bec) or Mtfn2 siRNAs and incubated with RC 12 , were stained for TOMM20 and confocal Z- stacks were acquired (three independent experiments). Scale bar: 10 µm ( i ). Fraction of RC 12 overlapping TOMM20 staining ( j ). siCtrl ( n = 17), siBec ( n = 18), and siMtfn2 ( n = 13, unpaired t -test). k Measurement of OCR in MOLM14 cells transfected with Ctrl ( n = 8), Bec ( n = 7), or Mtfn2 siRNAs ( n = 8) ± Oleate-BSA. l – o MOLM14 cells were transduced with the mitochondria–ER organelle linker (OMM-ER) and its control (OMM), treated ± metformin, and subjected to Western blot analysis for actin and LC3B ( l , m ) or stained for Bodipy and DAPI ( n , o ) (three independent experiments). Scale bar: 10 µm. Histograms represent the number of LC3B puncta ( m ) or the area of Bodipy dots per cell ( o ) unpaired t -test. Data are means ± s.e.m.

Article Snippet: Mouse antibodies from Santa Cruz Biotechnology against IP3R1 (sc-271197) and E2F1 (KH95), from Millipore against actin, rabbit antibody from Abcam against VDAC1 (ab15865), from PROGEN against ADRP (610102), from R&D System against FABP4 (AF3150), from Atlas Antibodies against ACLY (HPA022434), from Novus Biologicals against SREPB1 (NB600-582), from Invitrogen against SREBP2 (PA1-338), and from GeneTex against TOMM20 (GTX133756) were also used.

Techniques: Transfection, Control, Electron Microscopy, Proximity Ligation Assay, Transduction, Staining, Incubation, Western Blot

Journal: Nature Communications

Article Title: Autophagy regulates fatty acid availability for oxidative phosphorylation through mitochondria-endoplasmic reticulum contact sites

doi: 10.1038/s41467-020-17882-2

Figure Lengend Snippet: a , b NSG mice ( n = 31) were engrafted with MOLM14 cells expressing the Ctrl or VDAC1 shRNAs by intravenous injection. Seventeen days post graft, five mice shCtrl and six mice shVDAC1 were killed, and the number of human cells (hCD45+ and hCD33+) in the bone marrow and spleen was analyzed by flow cytometry. Graphs represent the number of human positive cells for hCD45 and hCD33 within the murine bone marrow and spleen from one experiment ( a ) (unpaired t -test). The remaining mice per group were used for overall survival analysis ( b ). Graph represents the Kaplan–Meier survival curves (Log-rank test). c , d NSG mice ( n = 39) were engrafted with MOLM14 cells and daily treated with vehicle or IACS-010759 by gavage. Seventeen days post graft, nine mice vehicle group and 11 mice IACS-010759-treated group were killed, and the number of human cells (hCD45+ and hCD33+) in the bone marrow and spleen was analyzed by flow cytometry. Graphs represent the number of human positive cells for hCD45 and hCD33 within the murine bone marrow and spleen ( c ) (unpaired t -test). The remaining mice were used for overall survival analysis ( d ). Graph represents the Kaplan–Meier survival curves (Log-rank test). e – g Viable bone marrow AML blasts from mice treated with IACS-010759 were stained for Bodipy 493/503 and DAPI. Scale bar: 10 µm ( e ). Histograms show the number or the area of Bodipy 493/503 dots per cell ( f , g ) (unpaired t -test). h Viable bone marrow AML blasts from mice treated with IACS-010759 (one experiment) were subjected to Western blot analysis for actin and LC3B. Numbers indicate ratios obtained by densitometric analysis. i Purified viable bone marrow AML blasts from mice treated with IACS-010759 (one experiment) were subjected to PLA assay. Numbers represent the number of PLA dots per cell (minimum 130 cells, unpaired t -test). Scale bar: 10 µm ( j ). Schematic diagram depicting the interplay between the autophagy process and the mitochondria in AML cells. Autophagy appears as a major regulator of mitochondria activity with the mitochondria controlling its supply of FFAs by regulating the number of autophagosomes via the formation MERCs. Data are means ± s.e.m.

Article Snippet: Mouse antibodies from Santa Cruz Biotechnology against IP3R1 (sc-271197) and E2F1 (KH95), from Millipore against actin, rabbit antibody from Abcam against VDAC1 (ab15865), from PROGEN against ADRP (610102), from R&D System against FABP4 (AF3150), from Atlas Antibodies against ACLY (HPA022434), from Novus Biologicals against SREPB1 (NB600-582), from Invitrogen against SREBP2 (PA1-338), and from GeneTex against TOMM20 (GTX133756) were also used.

Techniques: Expressing, Injection, Flow Cytometry, Staining, Western Blot, Purification, Activity Assay

Journal: Molecular nutrition & food research

Article Title: Lack of β, β-carotene -9’, 10’-oxygenase 2 leads to hepatic mitochondrial dysfunction and cellular oxidative stress in mice

doi: 10.1002/mnfr.201600576

Figure Lengend Snippet: Functional categorization of proteins differentially expressed in hepatic mitochondria of BCO2 knockout (KO) vs. 129S6 wild type (WT) mice identified by spectrum counting. Values are means, n=3 mice/group with 4 technical replicates/mouse.

Article Snippet: Antibodies against fatty acid synthase (FASN) (catalog # 10624-2-Ap), ATP synthase α subunit 1 (ATP5A1) (catalog # 14676-1-AP), citrate synthase (catalog # 16131-1-AP), hydroxyacyl-coenzyme A dehydrogenase (HADH) (catalog # 19828-1-AP), succinate dehydrogenase α (SDHA) (catalog # , 14865-1-AP), and nicotinamide nucleotide transhydrogenase (NNT) (catalog # 13442-2-AP) were purchased from ProteinTech Group (Chicago, IL, USA); Antibodies against lysosome-associated membrane protein 1 (LAMP1) (catalog # sc-20011), catalase (catalog # sc-50508), NNT (catalog # sc-163154), glutathione reductase (GSR) (catalog # sc-133245) were purchased from Santa Cruz Biotech (Dallas, TX, USA); peroxisome proliferator-activated receptor α (PPARα) antibody (catalog # 101710) was purchased from Cayman (Ann Arbor, MI, USA); Antibodies against β-actin (catalog # 4967), phosphor-Thr172-AMP-activated protein kinase α (pT172-AMPKα) (catalog # 2535), and AMPKα (catalog # 2603) were purchased from Cell Signaling Technology (Danvers, MA, USA); voltage-dependent anion-selective channel protein 1 (VDAC1) antibody (catalog # PA1780) was purchased from Boster Biosciences (Pleasanton, CA, USA).

Techniques: Functional Assay, Knock-Out, Membrane

Journal: Brain Sciences

Article Title: Differential Expression of Multiple Disease-Related Protein Groups Induced by Valproic Acid in Human SH-SY5Y Neuroblastoma Cells

doi: 10.3390/brainsci10080545

Figure Lengend Snippet: Pathway enrichment.

Article Snippet: Cellular protein was mixed with sample buffer (62.5 mM Tris-HCl pH = 6.8, 2% SDS, 25% glycerol, 0.01% Bromophenol Blue, 5% β-mercaptoethanol) and denatured by heating at 95 °C for 10 min. Immunoblotting analysis was performed using standard protocols with the following primary antibodies: rabbit anti-ATP5D (A9929, ABclonal, Woburn, MA, USA); rabbit anti-ATP5J (tcea21587, Taiclone, Taipei, Taiwan); rabbit anti-DPH6 (23993-1-AP, Proteintech, Rosemont, IL, USA); rabbit anti-FBXO4 (tcea12933, Taiclone); rabbit anti-FSTL1 (A15789, ABclonal); mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (G8795, Sigma-Aldrich); mouse anti-HDAC1 (GTX100513, GeneTex, Hsinchu City, Taiwan); rabbit anti-HDAC2 (GTX112957, GeneTex); rabbit anti-HIST1H2B (MAB15119, Abnova, Taipei, Taiwan); rabbit anti-KCNAB2 (17890-1-AP, Proteintech); rabbit anti-NPTX2 (A12031, ABclonal); rabbit anti-SCG3 (A7799, ABclonal); rabbit anti-SMARCA4 (tcea559, Taiclone); rabbit anti-UBE1L (tcea10708, Taiclone); rabbit anti-UBE2D1 (tcea7105, Taiclone); rabbit anti-UQCRB (tcea18400, Taiclone); rabbit anti-VDAC1 (tcea521, Taiclone); rabbit anti-VSNL1 (A6999, ABclonal).

Techniques: Expressing

Journal: Brain Sciences

Article Title: Differential Expression of Multiple Disease-Related Protein Groups Induced by Valproic Acid in Human SH-SY5Y Neuroblastoma Cells

doi: 10.3390/brainsci10080545

Figure Lengend Snippet: Immunoblotting analysis to validate the differential expression for 9 proteins in VPA-treated and PBS-treated biological replicated SH-SY5Y cells. ( A ) Immunoblotting showed the expression of ATP5J, FBXO4, HDAC1, HDAC2, SMARCA4, UBE1L, UBE2D1, UQCRB, and VDAC17 in VPA-treated and PBS-treated SH-SY5Y cells. ( B ) Quantification showing the fold differences in the expression of ATP5J, FBXO4, HDAC1, HDAC2, SMARCA4, UBE2D1, UQCRB, and VDAC1 between VPA-treated groups and PBS control. GAPDH was a loading control. The data are expressed as fold change to PBS ± SD (* p < 0.05, n = 3 to 6).

Article Snippet: Cellular protein was mixed with sample buffer (62.5 mM Tris-HCl pH = 6.8, 2% SDS, 25% glycerol, 0.01% Bromophenol Blue, 5% β-mercaptoethanol) and denatured by heating at 95 °C for 10 min. Immunoblotting analysis was performed using standard protocols with the following primary antibodies: rabbit anti-ATP5D (A9929, ABclonal, Woburn, MA, USA); rabbit anti-ATP5J (tcea21587, Taiclone, Taipei, Taiwan); rabbit anti-DPH6 (23993-1-AP, Proteintech, Rosemont, IL, USA); rabbit anti-FBXO4 (tcea12933, Taiclone); rabbit anti-FSTL1 (A15789, ABclonal); mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (G8795, Sigma-Aldrich); mouse anti-HDAC1 (GTX100513, GeneTex, Hsinchu City, Taiwan); rabbit anti-HDAC2 (GTX112957, GeneTex); rabbit anti-HIST1H2B (MAB15119, Abnova, Taipei, Taiwan); rabbit anti-KCNAB2 (17890-1-AP, Proteintech); rabbit anti-NPTX2 (A12031, ABclonal); rabbit anti-SCG3 (A7799, ABclonal); rabbit anti-SMARCA4 (tcea559, Taiclone); rabbit anti-UBE1L (tcea10708, Taiclone); rabbit anti-UBE2D1 (tcea7105, Taiclone); rabbit anti-UQCRB (tcea18400, Taiclone); rabbit anti-VDAC1 (tcea521, Taiclone); rabbit anti-VSNL1 (A6999, ABclonal).

Techniques: Western Blot, Expressing

Journal: Brain Sciences

Article Title: Differential Expression of Multiple Disease-Related Protein Groups Induced by Valproic Acid in Human SH-SY5Y Neuroblastoma Cells

doi: 10.3390/brainsci10080545

Figure Lengend Snippet: RT-qPCR assay showing the expression of 14 genes ( ATP5F1A , ATP5PF , BAZ1B , BMS1 , FBXO2 , HDAC1 , HDAC2 , SDHA , SMARCA4 , UBA7 , UBE2D1 , UQCRB , VDAC1 , and WDR36 ) in VPA-treated and PBS-treated SH-SY5Y cells. The GAPDH gene was used as the endogenous gene for normalization. The data are expressed as fold change to PBS ± SD (* p < 0.05, n = 3 to 6).

Article Snippet: Cellular protein was mixed with sample buffer (62.5 mM Tris-HCl pH = 6.8, 2% SDS, 25% glycerol, 0.01% Bromophenol Blue, 5% β-mercaptoethanol) and denatured by heating at 95 °C for 10 min. Immunoblotting analysis was performed using standard protocols with the following primary antibodies: rabbit anti-ATP5D (A9929, ABclonal, Woburn, MA, USA); rabbit anti-ATP5J (tcea21587, Taiclone, Taipei, Taiwan); rabbit anti-DPH6 (23993-1-AP, Proteintech, Rosemont, IL, USA); rabbit anti-FBXO4 (tcea12933, Taiclone); rabbit anti-FSTL1 (A15789, ABclonal); mouse anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (G8795, Sigma-Aldrich); mouse anti-HDAC1 (GTX100513, GeneTex, Hsinchu City, Taiwan); rabbit anti-HDAC2 (GTX112957, GeneTex); rabbit anti-HIST1H2B (MAB15119, Abnova, Taipei, Taiwan); rabbit anti-KCNAB2 (17890-1-AP, Proteintech); rabbit anti-NPTX2 (A12031, ABclonal); rabbit anti-SCG3 (A7799, ABclonal); rabbit anti-SMARCA4 (tcea559, Taiclone); rabbit anti-UBE1L (tcea10708, Taiclone); rabbit anti-UBE2D1 (tcea7105, Taiclone); rabbit anti-UQCRB (tcea18400, Taiclone); rabbit anti-VDAC1 (tcea521, Taiclone); rabbit anti-VSNL1 (A6999, ABclonal).

Techniques: Quantitative RT-PCR, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Pink1/PARK2/mROS-Dependent Mitophagy Initiates the Sensitization of Cancer Cells to Radiation

doi: 10.1155/2021/5595652

Figure Lengend Snippet: Mitochondrial dysfunction caused by mROS from Sarm1-KR and IR. (a) Representative FCM pictures of MMP in HeLa cells stained by Rh123 and analyzed by FCM. (b) The changes of Na + K + -ATPases, Ca 2+ Mg 2+ -ATPase, and mito-respiratory complex I and III activities by biochemical assay after light exposure and IR. (c) WB analysis was performed to determine protein levels of VDAC1 in total- and mito-protein. β -Actin and HSP60 proteins were used for loading control. (d) From top to bottom, the changes of VDAC1/ β -actin and VDAC1/HSP60. Bars represent mean ± SD of triplicate measurements. ∗ P < 0.05 and ∗∗ P < 0.01, versus control; # P < 0.05, versus 4 Gy IR; and △ P < 0.05, versus light exposure.

Article Snippet: Anti-COX IV, anti- β -actin, and anti-GAPDH were purchased from Santa Cruz, CA, USA; anti-voltage-dependent anion channel 1 (VDAC1), anti-heat-shock protein 60 (HSP60), anti-Pink1, and anti-PARK2 were purchased from Bioworld Technology, Inc., USA; and anti-microtubule-associated protein 1 light chain 3 (LC3), anti-p62, and anti-Tom20 were purchased from Cell Signaling Technology, Danvers, MA, USA.

Techniques: Staining