rabbit anti mt nd3  (Novus Biologicals)

Journal: Frontiers in Pharmacology

Article Title: Pterostilbene in Combination With Mitochondrial Cofactors Improve Mitochondrial Function in Cellular Models of Mitochondrial Diseases

doi: 10.3389/fphar.2022.862085

Figure Lengend Snippet: Screening of compounds in galactose medium. Cells were initially seeded in DMEM high glucose. After 3 days, glucose medium was changed to galactose. Images were acquired right after changing the medium and after 72 h of incubation. In optimal conditions control and both mutant cell lines present a similar proliferation rate (A–C) . Control cells almost do not alter their growth rate (D) but mutant ND3 and NDUFAF6 cells are unable to survive (E and F) . Pterostilbene (1 μM) treatment does not affect control cells (J) but promotes the survival of mutant ND3 and NDUFAF6 cells in galactose medium (H and I) . In addition, cocktail CoC3 (1 μM Pterostilbene, 5 μM nicotinamide, 1 μM riboflavin, 1 μM thiamine, 1 μM biotin, 5 μM lipoic acid and 1 μM L -carnitine) significantly improves pterostilbene’s positive effects (J–L) . CoC3 without ptersotilbene was also examined, but no favorable effect could be observed on mutant fibroblast (N and O) , suggesting that pterostilbene is necessary for the positive effect of CoC3. The quantification of cellular proliferation is shown in . Scale bar = 40 μm.

Article Snippet: NDUFAF6 antibody (DPABH-03128) was purchased from Creative Diagnostics (New York, United States). mtND1 (MBS9402205) and mtND3 (MBS8551680) antibodies were purchased from MyBioSource (San Diego, CA, United States). mtHSP70 antibody (MA3-028), HSP60 antibody (MA3-012), Tau antibody (MN1000), MitoTracker Deep Red FM (M22426), Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (A-31572) and Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (A-21202) were purchased from Thermo Fisher (Waltham, MA, United States). eif2α (5324), P-eif2α (9721), mtND3 (82,933), anti-acetylated lysine 9441) antibodies were purchase from Cell Signaling (Danvers, MA, United States).

Techniques: Incubation, Control, Mutagenesis

Journal: Frontiers in Pharmacology

Article Title: Pterostilbene in Combination With Mitochondrial Cofactors Improve Mitochondrial Function in Cellular Models of Mitochondrial Diseases

doi: 10.3389/fphar.2022.862085

Figure Lengend Snippet: Effect of CoC3 on mitochondrial protein expression levels in control and mutant fibroblasts. Western blot analysis of the affected mutant protein in each cell line: mtND3 (A) , NDUFAF6 (B) , COX15 (C) , NDUFS1 (D) , COQ7 (E) , NDUFS4 (F) . VDAC was used as mitochondrial mass marker, mtND3 and mtCO2 as mitochondrial-encoded reference proteins and NDUFA9 and nCOX4 as nuclear-encoded mitochondrial reference proteins. Overall, all patients show alterations with respect to the control, which are partially reversed after CoC3 treatment. “C” stands for healthy control cells. CoC3 treatment: 1 μM Pterostilbene, 5 μM nicotinamide, 1 μM riboflavin, 1 μM thiamine, 1 μM biotin, 5 μM lipoic acid and 1 μM L -carnitine. Differences between patients are probably due to the high variability in mutation severity and their genetic background. A representative actin band is shown, although loading control was additionally checked for every Western blot. Band densitometry can be found in .

Article Snippet: NDUFAF6 antibody (DPABH-03128) was purchased from Creative Diagnostics (New York, United States). mtND1 (MBS9402205) and mtND3 (MBS8551680) antibodies were purchased from MyBioSource (San Diego, CA, United States). mtHSP70 antibody (MA3-028), HSP60 antibody (MA3-012), Tau antibody (MN1000), MitoTracker Deep Red FM (M22426), Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (A-31572) and Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (A-21202) were purchased from Thermo Fisher (Waltham, MA, United States). eif2α (5324), P-eif2α (9721), mtND3 (82,933), anti-acetylated lysine 9441) antibodies were purchase from Cell Signaling (Danvers, MA, United States).

Techniques: Expressing, Control, Mutagenesis, Western Blot, Marker

Journal: Frontiers in Pharmacology

Article Title: Pterostilbene in Combination With Mitochondrial Cofactors Improve Mitochondrial Function in Cellular Models of Mitochondrial Diseases

doi: 10.3389/fphar.2022.862085

Figure Lengend Snippet: Effect of CoC3 on complex I and complex IV activities in control and mitochondrial mutant fibroblasts. Complex I proteins’ activities were measured using Complex I Enzyme Activity Dipstick Assay Kit from Abcam, with the exception of mutant COX15. In this particular mutation complex IV was measured. Mitochondrial complex activity in mtND3 cells (A) , NDUFAF6 cells (B) , COX15 cells (C) , NDUFS1 cells (D) , COQ7 cells (E) , NDUFS4 cells (F) . Results are shown in the dipstick images both in colour (bluish for complex I and yellowish for complex IV) and black/white. Band intensity was quantified using the Image Lab software. “C” stands for healthy control cells. Fibroblasts were treated for 7 days with CoC3 treatment: 1 μM Pterostilbene, 5 μM nicotinamide, 1 μM riboflavin, 1 μM thiamine, 1 μM biotin, 5 μM lipoic acid and 1 μM l -carnitine. * = p < 0.01 between Control and mutant fibroblasts. a p < 0.01 between untreated and treated mutant fibroblasts. a.u. (Arbitrary unit).

Article Snippet: NDUFAF6 antibody (DPABH-03128) was purchased from Creative Diagnostics (New York, United States). mtND1 (MBS9402205) and mtND3 (MBS8551680) antibodies were purchased from MyBioSource (San Diego, CA, United States). mtHSP70 antibody (MA3-028), HSP60 antibody (MA3-012), Tau antibody (MN1000), MitoTracker Deep Red FM (M22426), Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (A-31572) and Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (A-21202) were purchased from Thermo Fisher (Waltham, MA, United States). eif2α (5324), P-eif2α (9721), mtND3 (82,933), anti-acetylated lysine 9441) antibodies were purchase from Cell Signaling (Danvers, MA, United States).

Techniques: Control, Mutagenesis, Activity Assay, Software

Journal: Frontiers in Pharmacology

Article Title: Pterostilbene in Combination With Mitochondrial Cofactors Improve Mitochondrial Function in Cellular Models of Mitochondrial Diseases

doi: 10.3389/fphar.2022.862085

Figure Lengend Snippet: Effect of CoC3 on mitostress bioenergetic assays in control and mitochondrial mutant cell lines. Mitochondrial respiration profile was measured with a Seahorse XFe24 analyzer. Fibroblasts were treated for 7 days with CoC3 treatment: 1 μM Pterostilbene, 5 μM nicotinamide, 1 μM riboflavin, 1 μM thiamine, 1 μM biotin, 5 μM lipoic acid and 1 μM l -carnitine. Mutations in figure panels: mtND3 (A) , NDUFAF6 (B) , COX15 (C) , NDUFS1 (D) , COQ7 (E) , NDUFS4 (F) . * = p < 0.01 between Control and mutant fibroblasts. a p < 0.01 between untreated and treated mutant fibroblasts. OCR, oxygen consumption rate.

Article Snippet: NDUFAF6 antibody (DPABH-03128) was purchased from Creative Diagnostics (New York, United States). mtND1 (MBS9402205) and mtND3 (MBS8551680) antibodies were purchased from MyBioSource (San Diego, CA, United States). mtHSP70 antibody (MA3-028), HSP60 antibody (MA3-012), Tau antibody (MN1000), MitoTracker Deep Red FM (M22426), Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (A-31572) and Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (A-21202) were purchased from Thermo Fisher (Waltham, MA, United States). eif2α (5324), P-eif2α (9721), mtND3 (82,933), anti-acetylated lysine 9441) antibodies were purchase from Cell Signaling (Danvers, MA, United States).

Techniques: Control, Mutagenesis

Journal: Frontiers in Pharmacology

Article Title: Pterostilbene in Combination With Mitochondrial Cofactors Improve Mitochondrial Function in Cellular Models of Mitochondrial Diseases

doi: 10.3389/fphar.2022.862085

Figure Lengend Snippet: Effect of CoC3 on the expression levels of UPR mt -related proteins in control and mitochondrial mutant cell lines. Western blot analysis of several UPR mt -related proteins: Eif2α and P-eif2α proteins as Integrated Stress Response (ISR) markers; ATF4, ATF5 and CHOP proteins as canonical UPR mt proteins; HSP60 and mtHSP70 proteins as chaperones; SIRT3 protein as main target of pterostilbene; Nrf2 protein as a modulator of mitochondrial antioxidant pathways; NAMPT enzyme as NAD + /NADH cell metabolism regulator. “C” stands for healthy control cells. Fibroblasts were treated for 7 days with CoC3 treatment: 1 μM Pterostilbene, 5 μM nicotinamide, 1 μM riboflavin, 1 μM thiamine, 1 μM biotin, 5 μM lipoic acid and 1 μM L -carnitine. Mutations in figure panels: mtND3 (A) , NDUFAF6 (B) , COX15 (C) , NDUFS1 (D) , COQ7 (E) , NDUFS4 (F) . A representative actin band is shown for all assays, although loading control was checked for every Western blot. Band densitometry is shown in .

Article Snippet: NDUFAF6 antibody (DPABH-03128) was purchased from Creative Diagnostics (New York, United States). mtND1 (MBS9402205) and mtND3 (MBS8551680) antibodies were purchased from MyBioSource (San Diego, CA, United States). mtHSP70 antibody (MA3-028), HSP60 antibody (MA3-012), Tau antibody (MN1000), MitoTracker Deep Red FM (M22426), Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (A-31572) and Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (A-21202) were purchased from Thermo Fisher (Waltham, MA, United States). eif2α (5324), P-eif2α (9721), mtND3 (82,933), anti-acetylated lysine 9441) antibodies were purchase from Cell Signaling (Danvers, MA, United States).

Techniques: Expressing, Control, Mutagenesis, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Pterostilbene in Combination With Mitochondrial Cofactors Improve Mitochondrial Function in Cellular Models of Mitochondrial Diseases

doi: 10.3389/fphar.2022.862085

Figure Lengend Snippet: Effect of CoC3 on SIRT3 activity in control and mutant cell lines. SIRT3 activity was determined in mutant mtND3 and NDUFAF6 cells. Total SIRT3 activity was evaluated using the SIRT3 Activity Assay Kit (Fluorometric) from Abcam (A) . Pure SIRT3 protein was used as positive control, and no-enzyme and no-NAD (SIRT3 cofactor) samples as negative controls. Fluorescence was measured using a POLARstar Omega plate reader. Western blot of total protein acetylation in mutant mtND3 (B) and mutant NDUFAF6 (C) fibroblasts. Protein acetylation was assessed in mitochondria, cytoplasm and nuclei fractions. VDAC was used as mitochondrial protein marker; Actin was used as cytoplasm protein marker; SIRT1 was used as nuclear protein marker. Band densitometry is shown in . Fibroblasts were treated for 7 days with CoC3 treatment: 1 μM Pterostilbene, 5 μM nicotinamide, 1 μM riboflavin, 1 μM thiamine, 1 μM biotin, 5 μM lipoic acid and 1 μM L -carnitine. * = p < 0.01 between untreated and treated fibroblasts.

Article Snippet: NDUFAF6 antibody (DPABH-03128) was purchased from Creative Diagnostics (New York, United States). mtND1 (MBS9402205) and mtND3 (MBS8551680) antibodies were purchased from MyBioSource (San Diego, CA, United States). mtHSP70 antibody (MA3-028), HSP60 antibody (MA3-012), Tau antibody (MN1000), MitoTracker Deep Red FM (M22426), Donkey anti-Rabbit IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 555 (A-31572) and Donkey anti-Mouse IgG (H + L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (A-21202) were purchased from Thermo Fisher (Waltham, MA, United States). eif2α (5324), P-eif2α (9721), mtND3 (82,933), anti-acetylated lysine 9441) antibodies were purchase from Cell Signaling (Danvers, MA, United States).

Techniques: Activity Assay, Control, Mutagenesis, Positive Control, Fluorescence, Western Blot, Marker