Journal: Cell metabolism

Article Title: Ejection of damaged mitochondria and their removal by macrophages ensure efficient thermogenesis in brown adipose tissue

doi: 10.1016/j.cmet.2022.02.016

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: PDHβ (MR205484L4V, Origene, Rockville, MD, USA), SOD2 (MR201642L2V, Origene, Rockville, MD, USA), PINK1 (sc-44599-V, Santa Cruz Biotechnolgies, Dallas, TX, USA) or empty vector (pLenti-C-mGFP-P2A-Puro) was inoculated at 10 multiplicities of infection (MOI) with 2 μg/mL polybrene reagent in complete growth medium and then incubated for 48 h. Cells were selected with 2 μg/mL puromycin for three passages and maintained in 1 μg/mL puromycin.

Techniques: Immunofluorescence, Recombinant, Cytometry, Purification, shRNA, Cell Culture, Lysis, XF Assay, Staining, Isolation, Transfection, Live Cell Imaging, Mouse Assay, Software, Expressing

Journal: Frontiers in Pharmacology

Article Title: Enhancing Fatty Acids Oxidation via L-Carnitine Attenuates Obesity-Related Atrial Fibrillation and Structural Remodeling by Activating AMPK Signaling and Alleviating Cardiac Lipotoxicity

doi: 10.3389/fphar.2021.771940

Figure Lengend Snippet: LCA inhibits cardiac oxidative stress and mitigates inflammation. Representative images and analysis of the subcellular localization of the oxidation products of (A,B) MitoSOX and (C,D) DHE. Red: oxidation products-staining. Scar: 10 μm n = 4. (E,F) Comparisons of serum MDA and SOD using commercially available kits among the groups. (G,H) Levels of atrial MDA and SOD normalized to tissue protein concentration. (I) Schematic diagram illustrating NRF2-related signals. (J,K) Localization of NRF2 in atria by confocal immune-cyto-chemical analysis: Blue: nucleus (DAPI); Red: NRF2-staining; Pink: merge of blue and red indicated nuclear localization of NRF2 (Arrow). Scar: 30 μm n = 4. (L,M) Representative images and quantitative analysis of anti-oxidative system involved protein expressions (NRF2 and SOD2) using Western blot. (N,O) Representative images and analysis of oxidative DNA damage by 8-OH-dG staining. Green: 8-OH-dG -staining; Blue: DAPI. Scar: 50 μm n = 4. (P,Q) Representative images and analysis of DNA damage by TUNEL staining. Red: TUNEL-staining; Blue: DAPI; Pink: merge. Scar: 50 μm n = 4. Comparisons of (R) serum LDH and serum (S) CK-MB using commercially available kits. n = 8. (T) Quantitative analysis of the expression of inflammation-related genes in the atria using RT-qPCR. (U) Representative images and quantitative analysis of expression and phosphorylation of NFκB using Western blot. One-way ANOVA with Bonferroni post-hoc test was used to compare data among groups. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01. STD, standard diet; HFD, high-fat diet; LCA, l -carnitine; DHE, dihydroethidium; MDA, molondialdehyde; SOD, superoxide dismutase; NRF2, nuclear erythroid 2 p45-related factor 2; p-, phoso-; SOD2, manganese superoxide dismutase, superoxide dismutase 2; 8-OH-Dg, 8-hydroxydeoxyguanosine; IL, interleukin; TNF-α, tumor necrosis factor-α; MCP-1, monocyte chemoattractant protein-1; NFκB, the nuclear factor kappa B; LDH, lactate dehydrogenase; CK-MB, creatine kinase-MB.

Article Snippet: The membranes were blocked with 5% skim milk and incubated with antibodies against total-AMPK (1:1,000; Cell Signaling Technology/CST, Massachusetts, United States), CD36 (1:1,000; Abcam), collagen I (1:1,000; Abcam), collagen III (1:1,000; Abcam), connexin-43 (1:200; Invitrogen), CPT1B (1:1,000; Proteintech), GLUT4 (1:500; Proteintech), NFκB (1:1,000; Proteintech), NRF2 (1:1,000; Proteintech), phoso-AMPK (Thr172; 1:1,000; CST), phoso-Akt (Ser473; 1:1,000; CST), phoso-NFκB (1:1,000; CST), PGC1α (1:1,000; Proteintech), SOD2 (1:1,000; Proteintech), TGF-β (1:1,000; Abcam), α-SMA (1:1,000; CST), β-actin (1:5,000; Proteintech).

Techniques: Staining, Protein Concentration, Western Blot, TUNEL Assay, Expressing, Quantitative RT-PCR, Phospho-proteomics

Journal: Frontiers in Pharmacology

Article Title: Enhancing Fatty Acids Oxidation via L-Carnitine Attenuates Obesity-Related Atrial Fibrillation and Structural Remodeling by Activating AMPK Signaling and Alleviating Cardiac Lipotoxicity

doi: 10.3389/fphar.2021.771940

Figure Lengend Snippet: Pharmacological inhibition of AMPK via CC attenuated LCA-conferred beneficial effects in palmitate-treated primary atrial cardiomyocytes. (A) Schematic diagram illustrating the cell isolation, culture and treatment. (B) FAO rate in different treatment groups. The measure of substrate utilization after 18°C unsaturated fatty acid (Oleate, 100 μM) addition was normalized with maximal O 2 consumption in Control cells. (C) Representative images and (D) quantitative analysis of the expression of FAO-related proteins using Western blot. (E) Cellular MDA concentrations among the groups. (F) Representative images and (G) quantitative analysis of the expression of oxidative stress-related proteins (NRFS and SOD2) and inflammation-related protein (NFκB) using Western blot. n = 3 or 4 each group. Two-way ANOVA with Bonferroni post-hoc test was used to compare data among groups. Data are expressed as mean ± SEM. * p < 0.05, ** p < 0.01. CC, Compound C; LCA, l -carnitine; PA, palmitate; FAO, fatty acids oxidation; MDA, molondialdehyde; NRF2, nuclear erythroid 2 p45-related factor 2; AMPK, AMP-activated protein kinase; CD36, FAT; CPT1B, carnitine palmitoyltransferase-1B; p-, phoso-; PGC1α, peroxisome proliferator-activated receptor γ coactivator1α; NRF2, Nuclear factor erythroid 2-related factor 2; NFκB, the nuclear factor kappa B; SOD2, manganese superoxide dismutase, superoxide dismutase 2.

Article Snippet: The membranes were blocked with 5% skim milk and incubated with antibodies against total-AMPK (1:1,000; Cell Signaling Technology/CST, Massachusetts, United States), CD36 (1:1,000; Abcam), collagen I (1:1,000; Abcam), collagen III (1:1,000; Abcam), connexin-43 (1:200; Invitrogen), CPT1B (1:1,000; Proteintech), GLUT4 (1:500; Proteintech), NFκB (1:1,000; Proteintech), NRF2 (1:1,000; Proteintech), phoso-AMPK (Thr172; 1:1,000; CST), phoso-Akt (Ser473; 1:1,000; CST), phoso-NFκB (1:1,000; CST), PGC1α (1:1,000; Proteintech), SOD2 (1:1,000; Proteintech), TGF-β (1:1,000; Abcam), α-SMA (1:1,000; CST), β-actin (1:5,000; Proteintech).

Techniques: Inhibition, Cell Isolation, Control, Expressing, Western Blot

Journal: Molecules

Article Title: Resveratrol Ameliorates Fibrosis in Rheumatoid Arthritis-Associated Interstitial Lung Disease via the Autophagy–Lysosome Pathway

doi: 10.3390/molecules27238475

Figure Lengend Snippet: Resveratrol treatment reversed the accumulation of P62 to enhance autophagy levels and attenuate fibrosis. ( A ) The mRNA level of LC3B was quantified. ( B , C ) The correlation between LC3B and FN1 and COL3A1 gene expression showed a stronger positive correlation. Spearman’s r and p values are indicated. ( D ) Levels of SOD2, HIF-1a, and LC3 in lung tissues were detected using Western blotting. ( E ) The immunoblotting data of LC3 II/I were quantified as described above. ( F , G ) The mRNA levels of P62 and BNIP3 were quantified. ( H ) The correlation between P62 and FN1 gene expression showed a stronger positive correlation. Spearman’s r and p values are indicated. ( I ) Levels of P62, p-P62(phospho-SQSTM1/p62(Ser349)), BNIP3, BECN1, and BCL2 in lung tissues were detected using Western blotting. ( J ) The relative intensity of P62 was quantified as described above using Western blotting. Values represent the mean ± SEM (n = 3–5). * p < 0.05 and ** p < 0.01 compared with the control group, and compared with the model group (CIA+BLM), # p < 0.05 and ## p < 0.01.

Article Snippet: Then, the membrane was sealed in 5% skim milk for 1.5 h. β-actin (Abcam, # ab8226), Collagen I (Arigo Biolaboratories, #ARG21965), HIF-1a (Proteintech, #20960-1-AP), LC3A/B (Cell signaling technology, #12741), P62 (Cell signaling technology, #16177S), Phospho-SQSTM1/p62(Ser349) (CST, #E7M1A), Bcl-2 (Abcam, #ab182858), BNIP3 (Santa Cruz Biotechnology, #sc-56167), BNIP3L (Proteintech, #12986-1-AP), BECN1 (Boster, #PB0014), and SOD2 (Boster, #BA4566) were used.

Techniques: Gene Expression, Western Blot, Control