Journal: Molecular medicine (Cambridge, Mass.)

Article Title: PI3K p85α/HIF-1α accelerates the development of pulmonary arterial hypertension by regulating fatty acid uptake and mitophagy.

doi: 10.1186/s10020-024-00975-9

Figure Lengend Snippet: Fig. 1 HIF-1α was closely associated with the Parkin/PINK1 axis and fatty acid metabolism-related factors in PAH patients. A Serum levels of HIF-1α were measured by ELISA. B Serum levels of Parkin and PINK1 were measured by ELISA. C Serum levels of FAS, ACC, and CD36 were measured by ELISA. *P < 0.05 vs. Normal. The statistical test was performed using unpaired Student’s t-test. D Spearman’s correlation analysis was performed to determine the correlation between HIF-1α, Parkin, PINK1, FAS, ACC, and CD36. *P < 0.05. ***P < 0.001. ****P < 0.0001

Article Snippet: The levels of HIF-1α, Parkin, PINK1, fatty acid synthetase (FAS), acetyl-CoA carboxylase (ACC), and CD36 in human or rat serum were measured according to the instructions of the following ELISA kits: Human HIF-1α ELISA kit (CSB-E12112h, CUSABIO Co., Ltd., Wuhan, China), Human Parkin ELISA Kit (JL11195, Shanghai Jianglai Industrial Limited by Share Ltd., Shanghai, China), Human PINK1 ELISA Kit (JL11175, Shanghai Jianglai Industrial Limited by Share Ltd.), Human/Rat FAS ELISA Kit (CSB-E04542h/CSB-E07324r, CUSABIO Co., Ltd.), Human/Rat ACC ELISA Kit (CSBEL001119HU, CUSABIO Co., Ltd./YJ059228, Shanghai Yuanju Biotechnology Center, Shanghai, China), and Human/Rat CD36 ELISA Kit (CSB-E10117h/CSBEL004927RA, CUSABIO Co., Ltd.).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Nutrients

Article Title: Silibinin Restores NAD + Levels and Induces the SIRT1/AMPK Pathway in Non-Alcoholic Fatty Liver

doi: 10.3390/nu9101086

Figure Lengend Snippet: Effects of silibinin on de novo lipogenesis and lipolysis in HepG2. ( A – F ) HepG2 exposed to palmitate displayed lower levels of phosphorylation at the serine 79 residue of ACC, the rate-limiting enzyme of de novo lipogenesis. A 24 h culture with palmitate was also consistently associated with higher transcriptional activity of SREBP-1, the main transcription factor promoting lipogenesis, and higher levels of FAS, a downstream target of SREPB-1. Treatment with silibinin stimulated the phosphorylation of ACC, leading to its inactivation and thus to decreased conversion of acetyl-CoA to malonyl-CoA. It also reduced the transcriptional activity of SREBP-1, assessed on nuclear extracts, thus down-regulating the expression of FAS. Concomitantly with the effects on lipogenesis, we observed that treatment with silibinin up-regulated the expression of CPT1A, the rate limiting enzyme of mitochondrial β-oxidation. All values are expressed as mean ± SEM of three experiments ( n = 3) in duplicate. * p < 0.05 vs. vehicle; # p < 0.05 vs. FA. ACC: acetyl Co-A carboxylase; CPT1A: carnitine palmitoyltransferase 1A; FA: Fatty acid; FAS: Fatty acid synthase; PPARα: Peroxisome proliferator-activated receptor alpha; PPARδ: Peroxisome proliferator-activated receptor delta; SREBP-1: Sterol regulatory element-binding protein 1; SIL: Silibinin.

Article Snippet: Sandwich ELISA for threonine 172-phosphorylated AMPKα (#7959), total AMPKα (#7961), serine 79-phosphorylated acetyl-CoA carboxylase (ACC) (#7986), and total ACC (#7996) were purchased from Cell Signalling Technology (Beverly, MA, USA).

Techniques: Activity Assay, Expressing, Binding Assay

Journal: Nutrients

Article Title: Silibinin Restores NAD + Levels and Induces the SIRT1/AMPK Pathway in Non-Alcoholic Fatty Liver

doi: 10.3390/nu9101086

Figure Lengend Snippet: Effects of silibinin on hepatic de novo lipogenesis and lipolysis in mice. ( A – D ) Levels of phosphorylated and total ACC, the rate-limiting enzyme of de novo lipogenesis, were measured by sandwich ELISA and the ratio is showed. Phosphorylation at the serine 79 residue inhibits ACC activity, leading to decreased conversion of acetyl-CoA to malonyl-CoA. Following a 16-week HFD, mice displayed lower levels of hepatic phospho-ACC and higher levels of FAS, a main lipogenic enzyme. Treatment with silibinin induced ACC phosphorylation at Ser79 and down-regulated FAS expression. Consistently with these results, gene expression of CPT1A, the rate limiting enzyme of mitochondrial β-oxidation, and gene expression of PPARα, its upstream transcription factor, were downregulated in the livers of obese mice. Concomitantly with the effects on lipid accumulation, we observed that treatment with silibinin up-regulated the expression of PPARα and of CPT1A. All values are expressed as mean ± SEM of three experiments ( n = 3) in duplicate. * p < 0.05 vs. SD; # p < 0.05 vs. HFD. ACC: Acetyl-coA carboxylase; CPT1A: Carnitine O-palmitoyltransferase 1A; FAS: Fatty acid synthase; HFD: High fat diet; PPARα: Peroxisome proliferator-activated receptor alpha; SD: Standard diet; SIL: Silibinin.

Article Snippet: Sandwich ELISA for threonine 172-phosphorylated AMPKα (#7959), total AMPKα (#7961), serine 79-phosphorylated acetyl-CoA carboxylase (ACC) (#7986), and total ACC (#7996) were purchased from Cell Signalling Technology (Beverly, MA, USA).

Techniques: Sandwich ELISA, Activity Assay, Expressing

Journal: Acta neuropathologica communications

Article Title: BCKDK loss impairs mitochondrial Complex I activity and drives alpha-synuclein aggregation in models of Parkinson's disease.

doi: 10.1186/s40478-024-01915-8

Figure Lengend Snippet: Fig. 1 BCKDK is decreased in DA neurons in patients and αSyn mouse models of PD. (a) Total lysates from the midbrains of 8-month wild type (WT) or A53T-αSyn expressing mice were assessed via Western blot (WB) using anti-BCKDK and anti-β-Actin (labeled Actin) antibodies. The density of BCKDK versus actin is quantitated and shown in histogram. n = 3 mice/group. (b) Total lysates from the midbrains of 12-month WT and A53T-αSyn mice were subjected to WB analysis; quantifications indicate ratio of the density of BCKDK signal to that of β-Actin and are shown in histogram. n = 3 mice/group. (c) DA neuronal SH-SY5Y cells stably expressing either myc, WT- αSyn-myc, or A53T-αSyn-myc were lysed and assessed using WB with the anti-BCKDK and anti-β-Actin antibodies. Data was quantified using blot intensities (normalized to β-Actin) from 6 independent experiments. (d) Acetyl CoA levels were assessed in mouse brain midbrains and cell samples using an Acetyl CoA ELISA kit. (e) Brain sections of 12-month WT and A53T-αSyn mice were stained with anti-BCKDK and anti-TH antibodies. n = 3 mice/group; scale bar: 100 μm. Average immunodensity of BCKDK in TH-expressing cells is plotted for each sample. (f) Brain sections from postmortem brain samples obtained from normal subjects and PD patients were stained using anti-BCKDK and anti-TH antibodies. BCKDK immunodensity per TH+ cell was quantified. n = 3 patients/group; scale bar: 100 μm. All data shown are mean ± SEM; all data were analyzed using either the Student’s t test or One-way ANOVA

Article Snippet: Lysates were centrifuged for 10 min at 1500 g, and kit instructions (A-CoA ELISA kit; Elabscience; E-EL-0125) were used to measure Acetyl CoA levels.

Techniques: Expressing, Western Blot, Labeling, Stable Transfection, Enzyme-linked Immunosorbent Assay, Staining

Journal: Acta neuropathologica communications

Article Title: BCKDK loss impairs mitochondrial Complex I activity and drives alpha-synuclein aggregation in models of Parkinson's disease.

doi: 10.1186/s40478-024-01915-8

Figure Lengend Snippet: Fig. 2 BCKDK deficiency induces mitochondrial damage and αSyn oligomerization. (a) BCKDK knockdown stable SH-SY5Y cell lines were harvested and subjected to Acetyl CoA ELISA assay. n = 3 independent experiments. (b) Cell viability was assessed in stable BCKDK knockdown SH-SY5Y cells using LDH assay. n = 3 independent experiments. (c) Cell proliferation was assessed in stable BCKDK knockdown cells using MTT assay. n = 7 independent experi ments. (d) Mitochondrial oxidative stress was measured using the MitoSOX fluorescent dye, a superoxide indicator. MPP+ was added where indicated at a concentration of 1mM for 48 h prior to MitoSOX treatment. Cells were imaged and fluorescence intensity was quantified for each group. n = 4 indepen dent experiments. (e) Mitochondrial membrane potential was assessed using TMRM fluorescent dye, which localizes to mitochondria with intact mem brane potentials. MPP+ was added at a concentration of 1mM for 48 h prior to TMRM treatment; fluorescence intensity was quantified. n = 4 independent experiments. (f) Blue Native PAGE was conducted with lysates obtained from mitochondrial fractions of control and BCKDK knockdown SH-SY5Y cells. Complex I was detected using an anti-NDUFS6 antibody. Complex I levels were normalized to LonP1 protein levels, which were measured using SDS- PAGE. n = 3 independent experiments. (g) Complex I activity was assessed using a Complex I enzyme activity assay kit. n = 3 independent experiments. (h) Control and BCKDK knockdown cells were transfected with αSyn tagged with N- or C-terminus Venus fluorescent protein fragments for 48 h. Cells were imaged and fluorescence intensity per expressing cell was quantified. n = 4 independent experiments. All data are mean ± SEM with at least three independent repeats. Data were analyzed using either Student’s t test or One-way ANOVA

Article Snippet: Lysates were centrifuged for 10 min at 1500 g, and kit instructions (A-CoA ELISA kit; Elabscience; E-EL-0125) were used to measure Acetyl CoA levels.

Techniques: Knockdown, Enzyme-linked Immunosorbent Assay, Lactate Dehydrogenase Assay, MTT Assay, Concentration Assay, Fluorescence, Membrane, Blue Native PAGE, Control, SDS Page, Activity Assay, Enzyme Activity Assay, Transfection, Expressing

Journal: Nature Communications

Article Title: Repressed Blautia -acetate immunological axis underlies breast cancer progression promoted by chronic stress

doi: 10.1038/s41467-023-41817-2

Figure Lengend Snippet: a Percentage of 4T1 cell death after co-culture with CD8 + T cells with or without NaAc ( n = 5 per group). b Percentage of CD8 + T cell death after co-culture with 4T1 cells with or without NaAc ( n = 5 per group). c Quantification of IFN-γ abundance in CD8 + T cells after NaAc treatment under co-culture with 4T1 cancer cells by flow cytometry ( n = 5) or ELISA kit ( n = 6). d Heatmap (left panel) and MA-plot (right panel) of control and acetate-exposed CD8 + T cells under co-culture with 4T1 cell transcriptome ( n = 5 per group). e Pathway of acetate uptake and metabolism in CD8 + T cells. Created with BioRender.com. f mRNA expression of the solute carrier receptors Slc16a1 , Slc16a3 , Acss1 , Acss2 , and Acly in control and acetate-exposed CD8 + T cells under co-culture with 4T1 cells as determined by qPCR ( n = 4 per group). g Metabolic isotopic tracing analysis of CD8 + T cells under co-culture with 4T1 cells after exposure to 13 C-acetate. The x -axis shows the number of 13 C per respective metabolite. Depicted are pooled data from two independent experiments with cells from n = 3 mice each. h Acetyl-CoA concentration in CD8 + T cell lysates after co-culture with 4T1 cells with or without acetate ( n = 5 per group). Data were presented as mean ± SEM. Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test for ( a – c ) and unpaired two-tailed Student’s t -test for ( f – h ). Significance levels are denoted as * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. “ns” means no significant difference. Source data and exact p values are provided in the Source data file.

Article Snippet: The concentration of acetyl-CoA in cell lysates was measured by using the CUSABIO mouse Acetyl-CoA ELISA kit, according to the manufacturer’s instructions.

Techniques: Co-Culture Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Control, Expressing, Concentration Assay, Two Tailed Test