Journal: Oxidative medicine and cellular longevity

Article Title: Mitochondrial-Targeting Antioxidant SS-31 Suppresses Airway Inflammation and Oxidative Stress Induced by Cigarette Smoke.

doi: 10.1155/2021/6644238

Figure Lengend Snippet: Figure 6: SS-31 prevents mitochondrial dysfunction in CS-induced mouse lung. (a–d) Protein levels of OPA1, MFF (a, b), and cytochrome c (c, d) in lungs were evaluated by western blot following exposure to CS with or without SS-31 pretreatment. GAPDH or COX IV was used as a loading control (n = 3 per group). ∗∗P < 0:01 and ∗∗∗P < 0:001 vs. control; #P < 0:05 vs CS. Abbreviations: COX IV: cytochrome c oxidase IV; CS: cigarette smoke; Cyt c: cytochrome c; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; MFF: mitochondrial fission factor; mit: mitochondrion; OPA1: optic atrophy 1; SS-31 (H): high-dose SS-31 (5 mg/kg); SS-31 (L): low-dose SS-31 (2.5 mg/kg).

Article Snippet: After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membrane was incubated overnight at 4°C with antibodies against optic atrophy (OPA) 1 (mouse antibody from Cell Signaling Technology; human antibody from Proteintech, Rosemont, IL, USA); mitochondrial fission factor (MFF) (mouse antibody from Cell Signaling Technology; human antibody from Proteintech); and extracellular signal-regulated kinase (ERK), phosphorylated- (P-) ERK, P38, P-P38, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (all from Cell Signaling Technology).

Techniques: Western Blot, Control

Journal: Oxidative medicine and cellular longevity

Article Title: Mitochondrial-Targeting Antioxidant SS-31 Suppresses Airway Inflammation and Oxidative Stress Induced by Cigarette Smoke.

doi: 10.1155/2021/6644238

Figure Lengend Snippet: Figure 11: SS-31 protects against CSE-induced mitochondrial dysfunction via suppression of MAPK signaling. (a, b) Protein levels of OPA1 and MFF detected by western blot in BEAS-2B cells (n = 3 per group). ∗∗P < 0:01, ∗∗∗P < 0:001, and ∗∗∗∗P < 0:0001 vs. control; #P < 0:05 and ##P < 0:01 vs. ani+CSE; &P < 0:05 vs. CSE. Abbreviations: ani: anisomycin; CSE: cigarette smoke extract; MFF: mitochondrial fission factor; OPA1: optic atrophy 1.

Article Snippet: After blocking with 5% bovine serum albumin in Trisbuffered saline at room temperature for 1 h, the membrane was incubated overnight at 4°C with antibodies against optic atrophy (OPA) 1 (mouse antibody from Cell Signaling Technology; human antibody from Proteintech, Rosemont, IL, USA); mitochondrial fission factor (MFF) (mouse antibody from Cell Signaling Technology; human antibody from Proteintech); and extracellular signal-regulated kinase (ERK), phosphorylated- (P-) ERK, P38, P-P38, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (all from Cell Signaling Technology).

Techniques: Western Blot, Control

Journal: Frontiers in immunology

Article Title: The neonatal Fc receptor expression during macrophage differentiation is related to autophagy.

doi: 10.3389/fimmu.2022.1054425

Figure Lengend Snippet: FIGURE 3 FcRn mRNA and differentiation marker expression after FcRn siRNA transfection. M-Mf and GM-Mf were transfected with FcRn siRNA on day 3 for 24 h. (A) Transfection efficiency was assessed by RT-qPCR. Relative expression of FCGRT was normalized to that of actin and GAPDH. Data are relative FcRn levels to siRNA control (mean ± SD, N=7). (B) Flow cytometry of CD163, CD14, CD206 and CD16 expression after transfection in M-Mf and GM-Mf. Data are normalized MFI fold change to siRNA control (mean ± SD, N=7). M-Mf, M-CSF–induced macrophages; GM-Mf, GM-CSF–induced macrophages; MFI, mean fluorescence intensity; ns, not significant. ns = not significant and *p ≤0.05.

Article Snippet: An amount of 20 mMFcRn siRNA (Origene, USA) or siRNA control (Eurogentec, Belgium) was diluted in 200 ml Opti-MEM (Gibco, France) containing 1 ml Lipofectamine RNAiMAX (Thermo Fisher Scientific, USA).

Techniques: Marker, Expressing, Transfection, Quantitative RT-PCR, Control, Flow Cytometry

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Epigenetic Regulation of Hepatic Lipid Metabolism by DNA Methylation.

doi: 10.1002/advs.202206068

Figure Lengend Snippet: Figure 9. Regulation of DNMT1 protein stability by UBE3A. A) The association of the peaks of the chromatin accessibility at the Ube3a promoter analyzed by ATAC-seq and the reads of the Ube3a mRNA expression analyzed by RNA-seq in the liver of HFD-fed or LFD-fed mice. B) Immunoblotting analysis of UBE3A in the liver of mice fed LFD or HFD. All data are expressed as mean ± SEM. n = 5; *p < 0.05 versus LFD. C) Immunoblotting analysis of DNMT1 and DNMT3A protein in the liver of mice infected with AAV Ube3a expression virus. All data are expressed as mean ± SEM. n = 5; *p < 0.05 versus control. D) UBE3A regulation of DNMT1 ubiquitination. Ube3a was knocked down by shRNA or re-expressed into the knockdown cells with Ube3a expression vectors or overexpressed with Ube3a expression vectors in HEK293 cells. DNMT1 protein was immunoprecipitated with an anti-DNMT1 antibody and followed by immunoblotting with an anti-ubiquitin antibody. E) UBE3A promotes DNMT1 protein degradation in a cycloheximide (CHX) chase assay. The CHX chase assay was conducted in the HEK293 cells with Ube3a knockdown or overexpression. The cells were pre-treated with CHX and then harvested in a time course ranging from 0 to 16 h.

Article Snippet: For the models with liver Tet1, Tet2, or Tet3 knockdown, 7-week-old male C57BL/6J mice were intravenously injected with AAV8 Tet1 or Tet2 or Tet3 shRNA purchased from Addgene (Tet1 shRNA 85 742, Tet2 shRNA 86 743, Tet3 shRNA 85 740, and Control 85 741).

Techniques: Expressing, RNA Sequencing, Western Blot, Infection, Virus, Control, Ubiquitin Proteomics, shRNA, Knockdown, Immunoprecipitation, Over Expression

Journal: Journal of Nanobiotechnology

Article Title: Human periodontal ligament stem cell sheets activated by graphene oxide quantum dots repair periodontal bone defects by promoting mitochondrial dynamics dependent osteogenic differentiation

doi: 10.1186/s12951-024-02422-7

Figure Lengend Snippet: The effects of GOQDs on mitochondrial morphology and the expression of proteins regulating mitochondrial dynamics in hPDLSCs. A After 7 days of GOQD treatment, the mitochondrial morphology of hPDLSCs was observed via TEM. B After 7 days of GOQD treatment, morphological changes in the mitochondrial network were observed in hPDLSCs using laser confocal microscopy (MitoTracker Green). C Mitochondrial skeleton images obtained through Fiji software (upper plot) and statistical analysis of mitochondrial length and the mitochondrial footprint (bottom plot) by the Kruskal‒Wallis test ( n ≥ 3). D , E , F , G Western blot analysis of the expression of mitochondrial fusion marker proteins (MFN1, MFN2, and OPA1) after GOQD treatment for 3 days ( A ) or 7 days ( C ). E and G Relative quantitative analyses of ( D ) and ( F ) ( n = 3), respectively. H , I , J , K ) Western blot analysis of the expression of mitochondrial fission marker proteins (DRP1, MFF, and FIS1) after GOQD treatment for 3 days ( H ) or 7 days ( J ). I and K Relative quantitative analysis of ( H ) and ( J ), respectively ( n = 3). The vehicle group was the solvent control group. NS P > 0.05, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: The membranes were blocked with QuickBlockTM Blocking Buffer and incubated with QuickBlockTM Primary Antibody Dilution Buffer (P0239, Beyotime) and diluted Osterix (ab209484; Abcam, USA), Runx2 (A11753; ABclonal, China), OPN (A21084; ABclonal), OCN (A20800; ABclonal), Col-1 (14695-1-AP; Proteintech, USA), MFN1 (A21293; ABclonal), MFN2 (A19678; ABclonal), OPA1 (66583-1-Ig; Proteintech), DRP1 (A21968; ABclonal), MFF (A8700; ABclonal), FIS1 (A19666; ABclonal) and β-actin (66009-1-Ig; Proteintech) antibodies at 4 °C overnight.

Techniques: Expressing, Confocal Microscopy, Software, Western Blot, Marker, Solvent, Control

Journal: Oxidative Medicine and Cellular Longevity

Article Title: 5-Azacytidine and Resveratrol Enhance Chondrogenic Differentiation of Metabolic Syndrome-Derived Mesenchymal Stem Cells by Modulating Autophagy

doi: 10.1155/2019/1523140

Figure Lengend Snippet: Evaluation of mitochondrial dynamics during chondrogenic differentiation. Prior to experiments, cells were pretreated with AZA/RES for 24 hours, then the experimental medium was replaced by osteogenic differentiation medium. Cells were cultured in that medium for five days and after were subjected to further analysis. Mitochondrial morphology was visualized using TEM (a). Mitochondria from ASC EMS were characterized by disarrayed cristae and vacuole formation. Furthermore, expression of FIS (b) and MFN (c) was evaluated using RT-PCR. Amount of MFF and MFN protein cell lysates was established with western blot (d). Abbreviations: Mt: mitochondria, ER: endoplasmic reticulum, V: vacuole. Results expressed as mean ± S.D. Statistical significance indicated as an asterisk ( ∗ ) when comparing the result to ASC EMS and as a number sign (#) when comparing to ASC CTRL . ∗ p < 0.05; ### and ∗∗∗ p < 0.001.

Article Snippet: Afterwards, the membranes were washed with TBST and incubated with primary antibody against β -actin (Sigma-Aldrich, A5441), mitofusin-1 (MFN-1) (Biorbyt, orb11040,), and mitochondrial fission factor (MFF) (Biorbyt orb325479) at a dilution of 1 : 500 for 2 hours followed by incubation with ALP-conjugated secondary antibodies.

Techniques: Cell Culture, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot