Journal: BMC Musculoskeletal Disorders

Article Title: The effect of Miya on skeletal muscle changes by regulating gut microbiota in rats with osteoarthritis through AMPK pathway

doi: 10.1186/s12891-024-08203-5

Figure Lengend Snippet: Primer sequences for RT-qPCR assay

Article Snippet: The membranes were incubated with anti-AMPK antibody (cat. no. 2532S, 1: 1000, CST, Danvers, MA USA), anti-cholinergic receptor nicotinic alpha 1 subunit (Chrna1) antibody (cat. no. 10613–1-AP, 1: 1000, Proteintech, Rosemont, IL, USA), anti- lactate dehydrogenase (Ldh) antibody (cat. no. 21799–1-AP, 1: 1000, Abcam, Cambridge, UK), anti-medium-chain acyl-CoA dehydrogenase (Mcad) antibody (cat. no. 55210–1-AP, 1: 1000, Proteintech), anti-myogenic differentiation antigen (Myod) antibody (cat. no. 18943–1-AP, 1: 1000, Proteintech), anti-cholinergic receptor nicotinic delta subunit (Chrnd) antibody (cat. no. ab233758, 1: 1000, Abcam), anti-phosphatidylinositol-3 kinase (PI3K) antibody (cat. no. 20584–1-AP, 1: 1000, Proteintech), anti-long-chain acyl CoA dehydrogenase (Lcad) antibody (cat. no. 17526–1-AP, 1: 1000, Proteintech), and anti-GAPDH antibody (cat. no. 60004–1-lg, 1: 10,000, Proteintech) at 4 ̊C overnight; and after washing, the secondary antibody (cat. no. 111–035-003 or 115–035-003, 1: 5000, Jackson ImmunoResearch, West Grove, PA, USA) was added, and then incubated at 37 ̊C for 2 h. Finally, the protein bands were visualized using an enhanced chemiluminescence luminescence (cat. no. P0018AS, Beyotime, Shanghai, China).

Techniques:

Journal: BMC Musculoskeletal Disorders

Article Title: The effect of Miya on skeletal muscle changes by regulating gut microbiota in rats with osteoarthritis through AMPK pathway

doi: 10.1186/s12891-024-08203-5

Figure Lengend Snippet: Effects of the AMPK inhibitor on the mRNA levels of associated genes in the MY-treated OA using real-time quantitative PCR. The mRNA expression levels of ( A ) AMPK , ( B ) PI3K , ( C ) AKT1 , ( D ) mTOR , ( E ) p70s6k , ( F ) Ldh , ( G ) Lcad , ( H ) Mcad , ( I ) Myod , ( J ) Murf-1 , ( K ) Chrna1 , ( L ) Chrnd in the different groups. * p < 0.05, compared with the OA + MY group

Article Snippet: The membranes were incubated with anti-AMPK antibody (cat. no. 2532S, 1: 1000, CST, Danvers, MA USA), anti-cholinergic receptor nicotinic alpha 1 subunit (Chrna1) antibody (cat. no. 10613–1-AP, 1: 1000, Proteintech, Rosemont, IL, USA), anti- lactate dehydrogenase (Ldh) antibody (cat. no. 21799–1-AP, 1: 1000, Abcam, Cambridge, UK), anti-medium-chain acyl-CoA dehydrogenase (Mcad) antibody (cat. no. 55210–1-AP, 1: 1000, Proteintech), anti-myogenic differentiation antigen (Myod) antibody (cat. no. 18943–1-AP, 1: 1000, Proteintech), anti-cholinergic receptor nicotinic delta subunit (Chrnd) antibody (cat. no. ab233758, 1: 1000, Abcam), anti-phosphatidylinositol-3 kinase (PI3K) antibody (cat. no. 20584–1-AP, 1: 1000, Proteintech), anti-long-chain acyl CoA dehydrogenase (Lcad) antibody (cat. no. 17526–1-AP, 1: 1000, Proteintech), and anti-GAPDH antibody (cat. no. 60004–1-lg, 1: 10,000, Proteintech) at 4 ̊C overnight; and after washing, the secondary antibody (cat. no. 111–035-003 or 115–035-003, 1: 5000, Jackson ImmunoResearch, West Grove, PA, USA) was added, and then incubated at 37 ̊C for 2 h. Finally, the protein bands were visualized using an enhanced chemiluminescence luminescence (cat. no. P0018AS, Beyotime, Shanghai, China).

Techniques: Real-time Polymerase Chain Reaction, Expressing

Journal: BMC Musculoskeletal Disorders

Article Title: The effect of Miya on skeletal muscle changes by regulating gut microbiota in rats with osteoarthritis through AMPK pathway

doi: 10.1186/s12891-024-08203-5

Figure Lengend Snippet: Effects of the AMPK inhibitor on the protein levels of the associated proteins in the MY-treated OA by western blot. A The protein bands of all the associated proteins visualized by western blot analysis. The protein expression levels of ( B ) AMPK, ( C ) PI3K, ( D ) Ldh, ( E ) Lcad, ( F ) Mcad, ( G ) Myod, ( H ) Chrna1, ( I ) Chrnd in the different groups. * p < 0.05, compared with the OA + MY group

Article Snippet: The membranes were incubated with anti-AMPK antibody (cat. no. 2532S, 1: 1000, CST, Danvers, MA USA), anti-cholinergic receptor nicotinic alpha 1 subunit (Chrna1) antibody (cat. no. 10613–1-AP, 1: 1000, Proteintech, Rosemont, IL, USA), anti- lactate dehydrogenase (Ldh) antibody (cat. no. 21799–1-AP, 1: 1000, Abcam, Cambridge, UK), anti-medium-chain acyl-CoA dehydrogenase (Mcad) antibody (cat. no. 55210–1-AP, 1: 1000, Proteintech), anti-myogenic differentiation antigen (Myod) antibody (cat. no. 18943–1-AP, 1: 1000, Proteintech), anti-cholinergic receptor nicotinic delta subunit (Chrnd) antibody (cat. no. ab233758, 1: 1000, Abcam), anti-phosphatidylinositol-3 kinase (PI3K) antibody (cat. no. 20584–1-AP, 1: 1000, Proteintech), anti-long-chain acyl CoA dehydrogenase (Lcad) antibody (cat. no. 17526–1-AP, 1: 1000, Proteintech), and anti-GAPDH antibody (cat. no. 60004–1-lg, 1: 10,000, Proteintech) at 4 ̊C overnight; and after washing, the secondary antibody (cat. no. 111–035-003 or 115–035-003, 1: 5000, Jackson ImmunoResearch, West Grove, PA, USA) was added, and then incubated at 37 ̊C for 2 h. Finally, the protein bands were visualized using an enhanced chemiluminescence luminescence (cat. no. P0018AS, Beyotime, Shanghai, China).

Techniques: Western Blot, Expressing

Journal: Journal of Translational Medicine

Article Title: Identification and experimental validation of KMO as a critical immune-associated mitochondrial gene in unstable atherosclerotic plaque

doi: 10.1186/s12967-024-05464-5

Figure Lengend Snippet: Flowchart of the analysis. WGCNA, Weighted gene co-expression network analysis; LASSO, Least absolute shrinkage and selection operator; SVM-RFE, Support vector machine-recursive feature elimination; GSVA, Gene Set Variation Analysis; ROC, Receiver Operating Characteristic; IHC, immunohistochemistry; RT-qPCR, Quantitative real time polymerase chain reaction; AUC, areas under the curve; NT5DC3 , 5’-nucleotidase domain containing 3; ACADL , acyl-CoA dehydrogenase long chain; SLC25A4 , solute carrier family 25 member4; ALDH1B1 , aldehyde dehydrogenase 1 family member B1; MAOB , mono amineoxidase B; KMO , kynurenine3-monooxygenase

Article Snippet: Following a 30-minute room temperature block with 3% bovine serum albumin (BSA), the sections were incubated overnight at 4 °C with primary antibodies, including KMO antibody (Proteintech, Illinois, USA), 5’-nucleotidase domain containing 3 ( NT5DC3 ) antibody (Bioss, Beijing, China), acyl-CoA dehydrogenase long chain ( ACADL ) antibody (Proteintech, Illinois, USA), solute carrier family 25 member 4 ( SLC25A4 ) antibody (Affinity, Wuhan, China), aldehyde dehydrogenase 1 family member B1 ( ALDH1B1 ) antibody (Proteintech, Illinois, USA), monoamine oxidase B ( MAOB ) antibody (Proteintech, Illinois, USA), and GAPDH antibody (Affinity, Wuhan, China).

Techniques: Expressing, Selection, Plasmid Preparation, Immunohistochemistry, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Journal of translational medicine

Article Title: Identification and experimental validation of KMO as a critical immune-associated mitochondrial gene in unstable atherosclerotic plaque.

doi: 10.1186/s12967-024-05464-5

Figure Lengend Snippet: Fig. 1 Flowchart of the analysis. WGCNA, Weighted gene co-expression network analysis; LASSO, Least absolute shrinkage and selection operator; SVM- RFE, Support vector machine-recursive feature elimination; GSVA, Gene Set Variation Analysis; ROC, Receiver Operating Characteristic; IHC, immunohisto chemistry; RT-qPCR, Quantitative real time polymerase chain reaction; AUC, areas under the curve; NT5DC3, 5’-nucleotidase domain containing 3; ACADL, acyl-CoA dehydrogenase long chain; SLC25A4, solute carrier family 25 member4; ALDH1B1, aldehyde dehydrogenase 1 family member B1; MAOB, mono amineoxidase B; KMO, kynurenine3-monooxygenase

Article Snippet: Following a 30-minute room temperature block with 3% bovine serum albumin (BSA), the sections were incubated overnight at 4 °C with primary antibodies, including KMO antibody (Proteintech, Illinois, USA), 5’-nucleotidase domain containing 3 (NT5DC3) antibody (Bioss, Beijing, China), acyl-CoA dehydrogenase long chain (ACADL) antibody (Proteintech, Illinois, USA), solute carrier family 25 member 4 (SLC25A4) antibody (Affinity, Wuhan, China), aldehyde dehydrogenase 1 family member B1 (ALDH1B1) antibody (Proteintech, Illinois, USA), monoamine oxidase B (MAOB) antibody (Proteintech, Illinois, USA), and GAPDH antibody (Affinity, Wuhan, China).

Techniques: Expressing, Selection, Plasmid Preparation, Immunohistochemistry, Quantitative RT-PCR, Real-time Polymerase Chain Reaction

Journal: Journal of translational medicine

Article Title: Identification and experimental validation of KMO as a critical immune-associated mitochondrial gene in unstable atherosclerotic plaque.

doi: 10.1186/s12967-024-05464-5

Figure Lengend Snippet: Fig. 6 Pearson correlation analysis. Correlation between SLC25A4 (A), NT5DC3 (B), ALDH1B1 (C), MAOB (D), KMO (E), and ACADL (F) and infiltrating immune cells was assessed. Dot size indicates the strength of correlation between genes and immune cells, with larger dots indicating stronger correlations. Dot colour represents the corresponding p-value, with greener colours indicating lower p-values. A significance threshold of P < 0.05 was considered statisti cally significant

Article Snippet: Following a 30-minute room temperature block with 3% bovine serum albumin (BSA), the sections were incubated overnight at 4 °C with primary antibodies, including KMO antibody (Proteintech, Illinois, USA), 5’-nucleotidase domain containing 3 (NT5DC3) antibody (Bioss, Beijing, China), acyl-CoA dehydrogenase long chain (ACADL) antibody (Proteintech, Illinois, USA), solute carrier family 25 member 4 (SLC25A4) antibody (Affinity, Wuhan, China), aldehyde dehydrogenase 1 family member B1 (ALDH1B1) antibody (Proteintech, Illinois, USA), monoamine oxidase B (MAOB) antibody (Proteintech, Illinois, USA), and GAPDH antibody (Affinity, Wuhan, China).

Techniques:

Journal: Journal of translational medicine

Article Title: Identification and experimental validation of KMO as a critical immune-associated mitochondrial gene in unstable atherosclerotic plaque.

doi: 10.1186/s12967-024-05464-5

Figure Lengend Snippet: Fig. 7 Immunohistochemistry staining was performed to preliminarily evaluate the expression of KMO (A), ACADL (B), ALDH1B1 (C), MAOB (D), NT5DC3 (E), and SLC25A4 (F) between samples from SAP, UAP and control groups. Statistical analysis was conducted on data from the control, SAP, and UAP groups, with a sample size (n) of 5 for each group. Significance levels were indicated as * for P < 0.05, ** for P < 0.01, and *** for P < 0.001. Scale bars represent 50 μm. HE, Hematoxylin and Eosin; SAP, stable plaques; UAP, unstable plaques; Control, macroscopically intact arterial tissues

Article Snippet: Following a 30-minute room temperature block with 3% bovine serum albumin (BSA), the sections were incubated overnight at 4 °C with primary antibodies, including KMO antibody (Proteintech, Illinois, USA), 5’-nucleotidase domain containing 3 (NT5DC3) antibody (Bioss, Beijing, China), acyl-CoA dehydrogenase long chain (ACADL) antibody (Proteintech, Illinois, USA), solute carrier family 25 member 4 (SLC25A4) antibody (Affinity, Wuhan, China), aldehyde dehydrogenase 1 family member B1 (ALDH1B1) antibody (Proteintech, Illinois, USA), monoamine oxidase B (MAOB) antibody (Proteintech, Illinois, USA), and GAPDH antibody (Affinity, Wuhan, China).

Techniques: Immunohistochemistry, Staining, Expressing, Control

Journal: Journal of translational medicine

Article Title: Identification and experimental validation of KMO as a critical immune-associated mitochondrial gene in unstable atherosclerotic plaque.

doi: 10.1186/s12967-024-05464-5

Figure Lengend Snippet: Fig. 8 RT-qPCR analysis was performed to measure the relative RNA expression levels of KMO, ACADL, ALDH1B1, MAOB, NT5DC3, and SLC25A4 between samples from SAP, UAP, and control groups. Significance levels were indicated as * for P < 0.05, ** for P < 0.01, and *** for P < 0.001

Article Snippet: Following a 30-minute room temperature block with 3% bovine serum albumin (BSA), the sections were incubated overnight at 4 °C with primary antibodies, including KMO antibody (Proteintech, Illinois, USA), 5’-nucleotidase domain containing 3 (NT5DC3) antibody (Bioss, Beijing, China), acyl-CoA dehydrogenase long chain (ACADL) antibody (Proteintech, Illinois, USA), solute carrier family 25 member 4 (SLC25A4) antibody (Affinity, Wuhan, China), aldehyde dehydrogenase 1 family member B1 (ALDH1B1) antibody (Proteintech, Illinois, USA), monoamine oxidase B (MAOB) antibody (Proteintech, Illinois, USA), and GAPDH antibody (Affinity, Wuhan, China).

Techniques: Quantitative RT-PCR, RNA Expression, Control