Journal: Molecular Medicine Reports

Article Title: Glucose, glutamine, lactic acid and α-ketoglutarate restore metabolic disturbances and atrophic changes in energy-deprived muscle cells

doi: 10.3892/mmr.2025.13562

Figure Lengend Snippet: Effect of starvation on protein synthesis and degradation. Cells were incubated in starvation medium for 1, 5, or 24 h. (A) The relative mRNA expression of Atg1 and Murf1 was quantified by reverse transcription-quantitative PCR. (B and C) Protein phosphorylation levels of AMPK and p70S6K, and the protein expression ratio of LC3II/LC3I after (B) 5, or (C) 24 h of starvation were analyzed by western blotting. Representative blot images are shown in the upper part. Values are expressed as fold-change compared with the values of control cells incubated in regular DMEM. Values are presented as the mean ± SEM (n=3). *P<0.05, **P<0.01, ***P<0.001 vs. control. Atg1, atrogin-1; Con, control; Murf1, muscle ring finger 1; p-, phosphorylated; Stv, starvation.

Article Snippet: The membranes were blocked with Blocking One-P (Nacalai Tesque) for 60 min at room temperature (22–24°C) and then incubated overnight (16–18 h) at 4°C with primary antibodies against each of the following proteins: LC3 (#2775), 70-kDa ribosomal protein S6 kinase (p70S6K; #9202), phosphorylated-p70S6K (#9205), AMP-activated protein kinase (AMPK; #2532), and phosphorylated-AMPK (#2535), all purchased from Cell Signaling (Danvers, MA, USA), or β-actin (#sc47778; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Incubation, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Phospho-proteomics, Western Blot, Control

Journal: Molecular Medicine Reports

Article Title: Glucose, glutamine, lactic acid and α-ketoglutarate restore metabolic disturbances and atrophic changes in energy-deprived muscle cells

doi: 10.3892/mmr.2025.13562

Figure Lengend Snippet: Effect of single nutrient supplementation on protein metabolism and morphological atrophy. Cells were incubated in starvation medium with (colored bars) or without (black bars) the indicated nutrients for (A) 5 or (B and C) 24 h. (A) Relative mRNA expression of Atg1 and Murf1 was quantified by reverse transcription-quantitative PCR. (B) Protein phosphorylation levels of AMPK and p70S6K, and the protein expression ratio of LC3II/LC3I were analyzed by western blotting. Representative blot images are shown on the left side. (C) Representative fluorescence images of MHC antibody-stained myotubes at ×200 magnification were captured using a fluorescence microscope (BZ-X700; Keyence). Values are expressed as fold-change compared with the values of the control cells incubated in regular DMEM. Values are presented as mean ± SEM (n=3-4). Δ P<0.1, *P<0.05, **P<0.01, ***P<0.001 vs. starved cells. Atg1, atrogin-1; Con, control; Murf1, muscle ring finger 1; p-, phosphorylated; Stv, starvation; Glc, glucose; Gln, glutamine; LA, lactate; βOHB, β-hydroxy butyric acid; αKG, α-ketoglutarate; Leu, leucine.

Article Snippet: The membranes were blocked with Blocking One-P (Nacalai Tesque) for 60 min at room temperature (22–24°C) and then incubated overnight (16–18 h) at 4°C with primary antibodies against each of the following proteins: LC3 (#2775), 70-kDa ribosomal protein S6 kinase (p70S6K; #9202), phosphorylated-p70S6K (#9205), AMP-activated protein kinase (AMPK; #2532), and phosphorylated-AMPK (#2535), all purchased from Cell Signaling (Danvers, MA, USA), or β-actin (#sc47778; Santa Cruz Biotechnology, Dallas, TX, USA).

Techniques: Incubation, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Phospho-proteomics, Western Blot, Fluorescence, Staining, Microscopy, Control

Journal: Translational Neurodegeneration

Article Title: Deficient AMPK-SENP1-Sirt3 signaling impairs mitochondrial complex I function in Parkinson’s disease model

doi: 10.1186/s40035-025-00489-2

Figure Lengend Snippet: MPTP/MPP + attenuates AMPK activity and diminishes mitochondrial translocation of SENP1. a Relative intensities of AMPK (total lysates), P-AMPK/AMPK (total lysates), SENP1 (total lysates), SENP1 (mitochondrial lysates) and SUMO1-conjugated proteins (mitochondrial lysates) in the ventral midbrain of 3-month-old WT mice injected with MPTP or saline. Data were from 3 biological replicates, and are expressed as mean ± SD, n = 3 mice. b Left, western blotting for SENP1 (total lysates), SENP1 (mitochondrial lysates), AMPK (total lysates) and P-AMPK (total lysates) from SH-SY5Y cells treated with culture medium, MPP + , or MPP + and metformin (2 mmol/L) for 24 h, as well as blotting for SUMO1 in anti-Sirt3 immunoprecipitant from SH-SY5Y cell mitochondrial lysates. The total levels of Sirt3, SUMO1-conjugated proteins, and acetyl-conjugated proteins in mitochondrial lysates were also detected. Right, relative intensities of AMPK (total lysates), P-AMPK/AMPK (total lysates), SENP1 (total lysates), SENP1 (mitochondrial lysates), SUMOylated Sirt3 to total Sirt3 (mitochondrial lysates), SUMO1-conjugated proteins (mitochondrial lysates) and acetyl-conjugated proteins (mitochondrial lysates) from 3 biological replicates. Data expressed as mean ± SD, n = 3. c SH-SY5Y cells were treated with culture medium, MPP + , or MPP + + metformin for 24 h. CCK8 assay was performed to measure cell viability. Data expressed as mean ± SD, n = 3. One-way ANOVA with Tukey's post-hoc test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: For Western blotting assays, the following primary antibodies were used: anti-GAPDH (1:1000; Cell Signaling, 5174; Danvers, MA), anti-ATP5A (1:300; Abcam, 110273; Cambridge, MA), anti-Sirt3 (1:1000; Cell Signaling, 5490), anti-NDUFS2 (1:4000; Abcam, 110249), anti-NDUFA5 (1:1000; Proteintech, 16640; Rosemont, IL), anti-NDUFS3 (1:2000; Abcam, 14711), anti-NDUFA9 (1:1000; Abcam, 14713), anti-NDUFV1 (1:2000; Proteintech, 11238), anti-NDUFS7 (1:2000; Proteintech, 15728), anti-SENP1 (1:1000; Abcam, 108981), anti-AMP-activated protein kinase (AMPK) (1:1000; Cell Signaling, 2532), anti-SUMO1 (1:1000, custom-made by Cheng's team) [ ], anti-phosphorylated AMPK (P-AMPK) (1:1000; Cell Signaling, 2535), anti-Pan-AcK (Acetyl-conjugated proteins; 1:1000; Cell Signaling, 9441), and anti-tyrosine hydroxylase (TH)(1:200; Abcam, 112).

Techniques: Activity Assay, Translocation Assay, Injection, Saline, Western Blot, CCK-8 Assay

Journal: Journal of Advanced Research

Article Title: Elevation of p53 sensitizes obese kidney to adriamycin-induced aberrant lipid homeostasis via repressing HNF4α-mediated FGF21 sensitivity

doi: 10.1016/j.jare.2024.07.014

Figure Lengend Snippet: p53-imparied lipid metabolism was restored by the exogenous supplementation of FGF21 . ( a ) The timeline of the experimental design shows the experimental processes performed. ( b - d ) Measurements of UACR, SCr, and BUN in mice from the different groups (n = 6). ( e ) Representative micrographs and quantifications of Oil Red O staining showing renal lipid deposition (n = 3). Scale bar: black 20 μm, red 10 μm. ( f ) The protein of SIRT1, p-AMPK, t-AMPK, SREBP-1c, and PPARα expression in each group and quantification of their levels (n = 6). ( g ) HK-2 cells were treated with PA plus ADR or in the presence or absence of Nutlin-3a (10 μM) and FGF21 for 24 h. Representative microphotographs of Oil Red O and Nile Red staining showing lipid deposition in HK-2 cells. Scale bar: black and white 20 μm. ( h ) HK-2 cells were treated with PA plus ADR or in the presence or absence of Nutlin-3a and FGF21 for 24 h. The protein expression of SIRT1, p-AMPK, t-AMPK, SREBP-1c, and PPARα in HK-2 cells was detected by western blot (n = 3). ( i ) Relative mRNA level of Srebp1c , Fas , Acc , Pparα , Mcad , Aco , and Cpt1 in HK-2 cells after the indicated treatments (n = 3). Data were presented as mean ± SD. * P < 0.05.

Article Snippet: The membranes were incubated with anti-NRF2 (1:1000, Abcam), anti-Kelch-like ECH-associated protein 1 (KEAP1), anti-HO-1 (1:1000, Proteintech), anti-NQO1 (1:1000, Santa Cruz), anti-SREBP-1c (1:1000, Affinity Biosciences, Cincinnati, OH, USA), anti-PPARα (1:1000, Affinity Biosciences), anti-p53 (1:1000, Proteintech), anti-FGF21 (1:1000, Abcam), anti-phosphorylated extracellular regulated protein kinases (p-ERK, 1:1000, Cell Signaling Technology), anti-ERK (1:1000, Cell Signaling Technology), anti-FGFR1 (1:1000, Cell Signaling Technology), anti-p-FGFR1 (1:1000, Abcam), anti-β-Klotho (KLB, 1:1000, ABclonal Technology), anti-hepatocyte nuclear factor 4 alpha (HNF4α, 1:1000, Abcam), anti-sirtuin 1 (SIRT1, 1:1000, Cell Signaling Technology), anti-phosphorylated AMPK (1:1000, Cell Signaling Technology), anti-AMPK (1:1000, Cell Signaling Technology), anti-β-Actin (1:1000, Servicebio), anti-Na + /K + -ATPase (1:1000, Cell Signaling Technology) and anti-Histone H3 (1:1000, Affinity Biosciences).

Techniques: Staining, Expressing, Western Blot

Journal: Journal of Advanced Research

Article Title: Elevation of p53 sensitizes obese kidney to adriamycin-induced aberrant lipid homeostasis via repressing HNF4α-mediated FGF21 sensitivity

doi: 10.1016/j.jare.2024.07.014

Figure Lengend Snippet: FGF21 mediated PFT-α induced alleviation of lipid accumulation depending on the SIRT1-AMPK pathway. ( a ) The expression and quantification of SIRT1, p-AMPK, and t-AMPK protein in each group (n = 6). ( b ) After transfecting with NC-siRNA or Fgf21 -siRNA for 24 h, HK-2 cells were treated with PA plus ADR and/or PFT-α for 24 h after pretreatment with or without recombinant human FGF21 (50 ng/ml for 2 h). The expression of SIRT1, p-AMPK, and t-AMPK protein in HK-2 cells were measured by western blot (n = 3). ( c ) HK-2 cells were treated with PA plus ADR or in the presence or absence of PFT-α and EX-527 (100 μM) for 24 h. The protein levels of SIRT1, p-AMPK, and t-AMPK in HK-2 cells were detected by western blot (n = 3). ( d ) After transfecting with NC-shRNA or Ampk -shRNA for 24 h, HK-2 cells were treated with PA and ADR and/or PFT-α for 24 h. The expression of SIRT1, p-AMPK, and t-AMPK protein in HK-2 cells were measured by western blot (n = 3). ( e ) Relative mRNA level of Srebp1c , Fas , Acc , Ppara , Mcad , Aco, and Cpt1 in HK-2 cells after the indicated treatments (n = 3). ( f ) The expression of SREBP-1c and PPARα protein in HK-2 cells after the indicated treatments and quantification of their levels (n = 3). Data were presented as mean ± SD. * P < 0.05.

Article Snippet: The membranes were incubated with anti-NRF2 (1:1000, Abcam), anti-Kelch-like ECH-associated protein 1 (KEAP1), anti-HO-1 (1:1000, Proteintech), anti-NQO1 (1:1000, Santa Cruz), anti-SREBP-1c (1:1000, Affinity Biosciences, Cincinnati, OH, USA), anti-PPARα (1:1000, Affinity Biosciences), anti-p53 (1:1000, Proteintech), anti-FGF21 (1:1000, Abcam), anti-phosphorylated extracellular regulated protein kinases (p-ERK, 1:1000, Cell Signaling Technology), anti-ERK (1:1000, Cell Signaling Technology), anti-FGFR1 (1:1000, Cell Signaling Technology), anti-p-FGFR1 (1:1000, Abcam), anti-β-Klotho (KLB, 1:1000, ABclonal Technology), anti-hepatocyte nuclear factor 4 alpha (HNF4α, 1:1000, Abcam), anti-sirtuin 1 (SIRT1, 1:1000, Cell Signaling Technology), anti-phosphorylated AMPK (1:1000, Cell Signaling Technology), anti-AMPK (1:1000, Cell Signaling Technology), anti-β-Actin (1:1000, Servicebio), anti-Na + /K + -ATPase (1:1000, Cell Signaling Technology) and anti-Histone H3 (1:1000, Affinity Biosciences).

Techniques: Expressing, Recombinant, Western Blot, shRNA

Journal: Journal of Advanced Research

Article Title: Elevation of p53 sensitizes obese kidney to adriamycin-induced aberrant lipid homeostasis via repressing HNF4α-mediated FGF21 sensitivity

doi: 10.1016/j.jare.2024.07.014

Figure Lengend Snippet: PFT-α or exogenous supplementation of FGF21 protected against diabetes-induced renal lipid accumulation. ( a ) The timeline of the experimental design shows the experimental processes performed. ( b ) Measurements of SCr and BUN in mice from each group (n = 6). ( c ) Measurements of blood glucose in each group (n = 6). ( d ) Representative images of sections of renal tissue stained with PAS staining and related quantitative analysis (n = 6). Scale bar: black 20 μm. ( e ) Representative photomicrographs and quantifications of Oil Red O staining showing lipid deposition in the kidney (n = 3). Scale bar: black 20 μm, red 10 μm. ( f ) The protein of SIRT1, p-AMPK, t-AMPK, SREBP-1c, and PPARα expression in each group and quantification of their levels (n = 6). ( g ) The timeline of the experimental design shows the experimental processes performed. ( h ) Measurements of SCr and BUN in mice from each group (n = 3). ( i ) Measurements of blood glucose in each group (n = 3). ( j ) Representative images of sections of renal tissue stained with PAS staining and related quantitative analysis (n = 3). Scale bar: black 20 μm. ( k ) Representative images and quantifications of Oil Red O staining showing lipid deposition (n = 3). Scale bar: black 20 μm, red 10 μm. ( l ) Protein expression and quantification of SIRT1, p-AMPK, t-AMPK, SREBP-1c, and PPARα by western blot in mice renal tissues of each group (n = 3). ( m ) HK-2 cells were treated with PFT-α or FGF21 in the presence of high glucose (30 μM) for 24 h. Representative microphotographs of Oil Red O and Nile Red staining showing lipid deposition in HK-2 cells. Scale bar: black and white 20 μm. ( n ) The protein of SIRT1, p-AMPK, t-AMPK, SREBP-1c, and PPARα expression in HK-2 cells after the indicated treatments and quantification of their levels (n = 3). Data were presented as mean ± SD. * P < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The membranes were incubated with anti-NRF2 (1:1000, Abcam), anti-Kelch-like ECH-associated protein 1 (KEAP1), anti-HO-1 (1:1000, Proteintech), anti-NQO1 (1:1000, Santa Cruz), anti-SREBP-1c (1:1000, Affinity Biosciences, Cincinnati, OH, USA), anti-PPARα (1:1000, Affinity Biosciences), anti-p53 (1:1000, Proteintech), anti-FGF21 (1:1000, Abcam), anti-phosphorylated extracellular regulated protein kinases (p-ERK, 1:1000, Cell Signaling Technology), anti-ERK (1:1000, Cell Signaling Technology), anti-FGFR1 (1:1000, Cell Signaling Technology), anti-p-FGFR1 (1:1000, Abcam), anti-β-Klotho (KLB, 1:1000, ABclonal Technology), anti-hepatocyte nuclear factor 4 alpha (HNF4α, 1:1000, Abcam), anti-sirtuin 1 (SIRT1, 1:1000, Cell Signaling Technology), anti-phosphorylated AMPK (1:1000, Cell Signaling Technology), anti-AMPK (1:1000, Cell Signaling Technology), anti-β-Actin (1:1000, Servicebio), anti-Na + /K + -ATPase (1:1000, Cell Signaling Technology) and anti-Histone H3 (1:1000, Affinity Biosciences).

Techniques: Staining, Expressing, Western Blot