Journal: Food Frontiers

Article Title: Miquelianin, a main functional flavonoid of lotus leaf, induces thermogenic signature via p38‐PINK1‐PARKIN‐mediated mitophagy and mimicking NRF2 signaling during brown adipocyte differentiation

doi: 10.1002/fft2.284

Figure Lengend Snippet: FIGURE 4 Miquelianin treatment (5 and 10 μM) regulates mitochondrial fission-fusion plasticity and suppresses PINK1-PARKIN-mediated mitophagy in C3H10T1/2 cells. (a) qPCR analysis of mitochondrial fission- and fusion-related genes. (b and c) Western blot analysis of fission- and fusion-related proteins (OPA1, FIS1, DRP1, MFN1, and MFN2). (d and e) The fluorescence images of mitophagy caused by FCCP (10 μM) in miquelianin-treated cells and the fluorescence intensity was analyzed by Image J, respectively. (f) qPCR analysis of mitophagy-related genes (PINK1, PRKN, BECLIN1, p62, LC3B, and LC3A). (g and h) Western blot analysis of mitophagy-related proteins (PINK1, PRKN, BECLIN1, p62, LC3B, and LC3A). (i and j) Immunofluorescence staining for BECLIN1 (×400) in miquelianin-treated C3H10T1/2 cells, and the fluorescence intensity was analyzed by Image J, respectively. Values with different letters determined by Duncan’s test indicate a significant difference (p < .05).

Article Snippet: Primary antibodies, including PKA (protein kinase A), p-PKA, CREB (cAMP response element binding protein), p-CREB, p38, p-p38, UCP1, ATGL (adipose triglyceride lipase), ABHD5 (α/β-Hydrolase domain-containing protein 5), PLIN5 (perilipin 5), SIRT1 (sirtuin 1), PGC-1α (peroxisome proliferatoractivated receptor-γ coactivator 1α), NRF2, TFAM (mitochondrial transcription factor A), COX-2 (cytochrome c oxidase subunit II), COX-IV, MFN1 (mitfusin1), MFN2, OPA1 (optic atrophy 1), DRP1 (dynamin-related protein 1), FIS1 (mitochondrial fission protein 1), PINK1, PARKIN, BECLIN1 (ATG6), LC-3B (microtubule-associated protein 1 light chain 3 beta), NAMPT (nicotinamide phosphoribosyltransferase) (Proteintech, 1:1000), and β3-AR (beta3-adrenergic receptor) (Abclonal, 1:1000) were incubated at 4◦C for 12 h. The HRPconjugated secondary antibodies (1:2000, Proteintech) were then employed for chemiluminescent detection with ECL detection reagent (Beyotime).

Techniques: Western Blot, Fluorescence, Immunofluorescence, Staining

Journal: Food Frontiers

Article Title: Miquelianin, a main functional flavonoid of lotus leaf, induces thermogenic signature via p38‐PINK1‐PARKIN‐mediated mitophagy and mimicking NRF2 signaling during brown adipocyte differentiation

doi: 10.1002/fft2.284

Figure Lengend Snippet: FIGURE 4 Miquelianin treatment (5 and 10 μM) regulates mitochondrial fission-fusion plasticity and suppresses PINK1-PARKIN-mediated mitophagy in C3H10T1/2 cells. (a) qPCR analysis of mitochondrial fission- and fusion-related genes. (b and c) Western blot analysis of fission- and fusion-related proteins (OPA1, FIS1, DRP1, MFN1, and MFN2). (d and e) The fluorescence images of mitophagy caused by FCCP (10 μM) in miquelianin-treated cells and the fluorescence intensity was analyzed by Image J, respectively. (f) qPCR analysis of mitophagy-related genes (PINK1, PRKN, BECLIN1, p62, LC3B, and LC3A). (g and h) Western blot analysis of mitophagy-related proteins (PINK1, PRKN, BECLIN1, p62, LC3B, and LC3A). (i and j) Immunofluorescence staining for BECLIN1 (×400) in miquelianin-treated C3H10T1/2 cells, and the fluorescence intensity was analyzed by Image J, respectively. Values with different letters determined by Duncan’s test indicate a significant difference (p < .05).

Article Snippet: Primary antibodies, including PKA (protein kinase A), p-PKA, CREB (cAMP response element binding protein), p-CREB, p38, p-p38, UCP1, ATGL (adipose triglyceride lipase), ABHD5 (α/β-Hydrolase domain-containing protein 5), PLIN5 (perilipin 5), SIRT1 (sirtuin 1), PGC-1α (peroxisome proliferatoractivated receptor-γ coactivator 1α), NRF2, TFAM (mitochondrial transcription factor A), COX-2 (cytochrome c oxidase subunit II), COX-IV, MFN1 (mitfusin1), MFN2, OPA1 (optic atrophy 1), DRP1 (dynamin-related protein 1), FIS1 (mitochondrial fission protein 1), PINK1, PARKIN, BECLIN1 (ATG6), LC-3B (microtubule-associated protein 1 light chain 3 beta), NAMPT (nicotinamide phosphoribosyltransferase) (Proteintech, 1:1000), and β3-AR (beta3-adrenergic receptor) (Abclonal, 1:1000) were incubated at 4◦C for 12 h. The HRPconjugated secondary antibodies (1:2000, Proteintech) were then employed for chemiluminescent detection with ECL detection reagent (Beyotime).

Techniques: Western Blot, Fluorescence, Immunofluorescence, Staining

Journal: Food Frontiers

Article Title: Miquelianin, a main functional flavonoid of lotus leaf, induces thermogenic signature via p38‐PINK1‐PARKIN‐mediated mitophagy and mimicking NRF2 signaling during brown adipocyte differentiation

doi: 10.1002/fft2.284

Figure Lengend Snippet: FIGURE 4 Miquelianin treatment (5 and 10 μM) regulates mitochondrial fission-fusion plasticity and suppresses PINK1-PARKIN-mediated mitophagy in C3H10T1/2 cells. (a) qPCR analysis of mitochondrial fission- and fusion-related genes. (b and c) Western blot analysis of fission- and fusion-related proteins (OPA1, FIS1, DRP1, MFN1, and MFN2). (d and e) The fluorescence images of mitophagy caused by FCCP (10 μM) in miquelianin-treated cells and the fluorescence intensity was analyzed by Image J, respectively. (f) qPCR analysis of mitophagy-related genes (PINK1, PRKN, BECLIN1, p62, LC3B, and LC3A). (g and h) Western blot analysis of mitophagy-related proteins (PINK1, PRKN, BECLIN1, p62, LC3B, and LC3A). (i and j) Immunofluorescence staining for BECLIN1 (×400) in miquelianin-treated C3H10T1/2 cells, and the fluorescence intensity was analyzed by Image J, respectively. Values with different letters determined by Duncan’s test indicate a significant difference (p < .05).

Article Snippet: Primary antibodies, including PKA (protein kinase A), p-PKA, CREB (cAMP response element binding protein), p-CREB, p38, p-p38, UCP1, ATGL (adipose triglyceride lipase), ABHD5 (α/β-Hydrolase domain-containing protein 5), PLIN5 (perilipin 5), SIRT1 (sirtuin 1), PGC-1α (peroxisome proliferatoractivated receptor-γ coactivator 1α), NRF2, TFAM (mitochondrial transcription factor A), COX-2 (cytochrome c oxidase subunit II), COX-IV, MFN1 (mitfusin1), MFN2, OPA1 (optic atrophy 1), DRP1 (dynamin-related protein 1), FIS1 (mitochondrial fission protein 1), PINK1, PARKIN, BECLIN1 (ATG6), LC-3B (microtubule-associated protein 1 light chain 3 beta), NAMPT (nicotinamide phosphoribosyltransferase) (Proteintech, 1:1000), and β3-AR (beta3-adrenergic receptor) (Abclonal, 1:1000) were incubated at 4◦C for 12 h. The HRPconjugated secondary antibodies (1:2000, Proteintech) were then employed for chemiluminescent detection with ECL detection reagent (Beyotime).

Techniques: Western Blot, Fluorescence, Immunofluorescence, Staining

Journal: Food Frontiers

Article Title: Miquelianin, a main functional flavonoid of lotus leaf, induces thermogenic signature via p38‐PINK1‐PARKIN‐mediated mitophagy and mimicking NRF2 signaling during brown adipocyte differentiation

doi: 10.1002/fft2.284

Figure Lengend Snippet: FIGURE 4 Miquelianin treatment (5 and 10 μM) regulates mitochondrial fission-fusion plasticity and suppresses PINK1-PARKIN-mediated mitophagy in C3H10T1/2 cells. (a) qPCR analysis of mitochondrial fission- and fusion-related genes. (b and c) Western blot analysis of fission- and fusion-related proteins (OPA1, FIS1, DRP1, MFN1, and MFN2). (d and e) The fluorescence images of mitophagy caused by FCCP (10 μM) in miquelianin-treated cells and the fluorescence intensity was analyzed by Image J, respectively. (f) qPCR analysis of mitophagy-related genes (PINK1, PRKN, BECLIN1, p62, LC3B, and LC3A). (g and h) Western blot analysis of mitophagy-related proteins (PINK1, PRKN, BECLIN1, p62, LC3B, and LC3A). (i and j) Immunofluorescence staining for BECLIN1 (×400) in miquelianin-treated C3H10T1/2 cells, and the fluorescence intensity was analyzed by Image J, respectively. Values with different letters determined by Duncan’s test indicate a significant difference (p < .05).

Article Snippet: Primary antibodies, including PKA (protein kinase A), p-PKA, CREB (cAMP response element binding protein), p-CREB, p38, p-p38, UCP1, ATGL (adipose triglyceride lipase), ABHD5 (α/β-Hydrolase domain-containing protein 5), PLIN5 (perilipin 5), SIRT1 (sirtuin 1), PGC-1α (peroxisome proliferatoractivated receptor-γ coactivator 1α), NRF2, TFAM (mitochondrial transcription factor A), COX-2 (cytochrome c oxidase subunit II), COX-IV, MFN1 (mitfusin1), MFN2, OPA1 (optic atrophy 1), DRP1 (dynamin-related protein 1), FIS1 (mitochondrial fission protein 1), PINK1, PARKIN, BECLIN1 (ATG6), LC-3B (microtubule-associated protein 1 light chain 3 beta), NAMPT (nicotinamide phosphoribosyltransferase) (Proteintech, 1:1000), and β3-AR (beta3-adrenergic receptor) (Abclonal, 1:1000) were incubated at 4◦C for 12 h. The HRPconjugated secondary antibodies (1:2000, Proteintech) were then employed for chemiluminescent detection with ECL detection reagent (Beyotime).

Techniques: Western Blot, Fluorescence, Immunofluorescence, Staining

Journal: Food Frontiers

Article Title: Miquelianin, a main functional flavonoid of lotus leaf, induces thermogenic signature via p38‐PINK1‐PARKIN‐mediated mitophagy and mimicking NRF2 signaling during brown adipocyte differentiation

doi: 10.1002/fft2.284

Figure Lengend Snippet: FIGURE 4 Miquelianin treatment (5 and 10 μM) regulates mitochondrial fission-fusion plasticity and suppresses PINK1-PARKIN-mediated mitophagy in C3H10T1/2 cells. (a) qPCR analysis of mitochondrial fission- and fusion-related genes. (b and c) Western blot analysis of fission- and fusion-related proteins (OPA1, FIS1, DRP1, MFN1, and MFN2). (d and e) The fluorescence images of mitophagy caused by FCCP (10 μM) in miquelianin-treated cells and the fluorescence intensity was analyzed by Image J, respectively. (f) qPCR analysis of mitophagy-related genes (PINK1, PRKN, BECLIN1, p62, LC3B, and LC3A). (g and h) Western blot analysis of mitophagy-related proteins (PINK1, PRKN, BECLIN1, p62, LC3B, and LC3A). (i and j) Immunofluorescence staining for BECLIN1 (×400) in miquelianin-treated C3H10T1/2 cells, and the fluorescence intensity was analyzed by Image J, respectively. Values with different letters determined by Duncan’s test indicate a significant difference (p < .05).

Article Snippet: Primary antibodies, including PKA (protein kinase A), p-PKA, CREB (cAMP response element binding protein), p-CREB, p38, p-p38, UCP1, ATGL (adipose triglyceride lipase), ABHD5 (α/β-Hydrolase domain-containing protein 5), PLIN5 (perilipin 5), SIRT1 (sirtuin 1), PGC-1α (peroxisome proliferatoractivated receptor-γ coactivator 1α), NRF2, TFAM (mitochondrial transcription factor A), COX-2 (cytochrome c oxidase subunit II), COX-IV, MFN1 (mitfusin1), MFN2, OPA1 (optic atrophy 1), DRP1 (dynamin-related protein 1), FIS1 (mitochondrial fission protein 1), PINK1, PARKIN, BECLIN1 (ATG6), LC-3B (microtubule-associated protein 1 light chain 3 beta), NAMPT (nicotinamide phosphoribosyltransferase) (Proteintech, 1:1000), and β3-AR (beta3-adrenergic receptor) (Abclonal, 1:1000) were incubated at 4◦C for 12 h. The HRPconjugated secondary antibodies (1:2000, Proteintech) were then employed for chemiluminescent detection with ECL detection reagent (Beyotime).

Techniques: Western Blot, Fluorescence, Immunofluorescence, Staining

Journal: Food Frontiers

Article Title: Miquelianin, a main functional flavonoid of lotus leaf, induces thermogenic signature via p38‐PINK1‐PARKIN‐mediated mitophagy and mimicking NRF2 signaling during brown adipocyte differentiation

doi: 10.1002/fft2.284

Figure Lengend Snippet: FIGURE 5 Miquelianin induces thermogenesis in C3H10T1/2 cells through β3-AR/PKA/p38-mediated mitophagy. (a–d) Docking model with the lowest binding affinity, total energy, root-mean-square deviation (RMSD), and radius of gyration (Rg) analysis, respectively. (e) The hydrogen bonds number in the complex during the simulation time. (f) 3D binding mode of the miquelianin-β3-AR complex. (g) 2D schematic interaction diagram between miquelianin and β3-AR. (h and i) Western blot analysis of PKA, p-PKA, p38, p-p38, CREB, and p-CREB in miquelianin (5 and 10 μM)-treated C3H10T1/2 cells. (j and k) Western blot analysis of PARKIN, BECLIN1, LC-3B, COX2, TFAM, and UCP1 in miquelianin (10 μM)-treated C3H10T1/2 cells stimulated with or without SB203580. Values with different letters determined by Duncan’s test indicate a significant difference (p < .05).

Article Snippet: Primary antibodies, including PKA (protein kinase A), p-PKA, CREB (cAMP response element binding protein), p-CREB, p38, p-p38, UCP1, ATGL (adipose triglyceride lipase), ABHD5 (α/β-Hydrolase domain-containing protein 5), PLIN5 (perilipin 5), SIRT1 (sirtuin 1), PGC-1α (peroxisome proliferatoractivated receptor-γ coactivator 1α), NRF2, TFAM (mitochondrial transcription factor A), COX-2 (cytochrome c oxidase subunit II), COX-IV, MFN1 (mitfusin1), MFN2, OPA1 (optic atrophy 1), DRP1 (dynamin-related protein 1), FIS1 (mitochondrial fission protein 1), PINK1, PARKIN, BECLIN1 (ATG6), LC-3B (microtubule-associated protein 1 light chain 3 beta), NAMPT (nicotinamide phosphoribosyltransferase) (Proteintech, 1:1000), and β3-AR (beta3-adrenergic receptor) (Abclonal, 1:1000) were incubated at 4◦C for 12 h. The HRPconjugated secondary antibodies (1:2000, Proteintech) were then employed for chemiluminescent detection with ECL detection reagent (Beyotime).

Techniques: Binding Assay, Western Blot