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: Cancer Science

Article Title: Human leukocyte antigen-E alleles and expression in patients with serous ovarian cancer

doi: 10.1111/cas.12641

Figure Lengend Snippet: Samples with different cytoplasmic staining intensities against human leukocyte antigen-E (HLA-E) protein by immunohistochemical analysis. (a) Negative HLA-E expression in the control tissue. (b) Weak HLA-E expression in serous ovarian cancer (SOC) with the HLA-E*0101*0101 genotype. (c) Moderate HLA-E expression in SOC with the HLA-E*0101*0103 genotype. (d) Strong HLA-E expression in SOC with the HLA-E*0103*0103 genotype. Bar = 50 μm.

Article Snippet: After boiling in 10 mmol/L of citrate buffer (pH 6.0) for antigen retrieval and cooling down, the sections were blocked with 1% BSA and incubated overnight at 4°C with primary mouse monoclonal anti-HLA-E antibody (MEM-E/02, 1:1000; Abcam, Cambridge, USA).

Techniques: Staining, Immunohistochemical staining, Expressing

Journal: Cancer Science

Article Title: Human leukocyte antigen-E alleles and expression in patients with serous ovarian cancer

doi: 10.1111/cas.12641

Figure Lengend Snippet: Expression levels of human leukocyte antigen-E (HLA-E) in transfected K562 cells. (a) Relative HLA-E mRNA expression was assessed using quantitative real-time PCR assay. The data were normalized to the gene expression of K562G using the standard method. (b) Examples of HLA-E protein intensity assayed by Western blot. (c) Expression level of HLA-E protein was normalized against that of GAPDH. (d) Examples of HLA-E cell surface expression detected by flow cytometry. Cells were stained with 3D12-APC mAb after incubation for 16 h at 26°C. Both HLA-E and EGFP positive cells represent living cells expressing HLA-E molecule on the cell surface. (e) Percentage of cells with HLA-E surface expression in the transfected cells. All results were expressed as mean ± SD for three replicate experiments. ** P < 0.01. K562G Control, K562 cells transfected with EGFP and pLL3.7; K562G-E*0101, K562 cells transfected with EGFP and pLL3.7-HLA-E ( HLA-E*0101 allele); K562G-E*0103, K562 cells transfected with EGFP and pLL3.7-HLA-E ( HLA-E*0103 allele).

Article Snippet: After boiling in 10 mmol/L of citrate buffer (pH 6.0) for antigen retrieval and cooling down, the sections were blocked with 1% BSA and incubated overnight at 4°C with primary mouse monoclonal anti-HLA-E antibody (MEM-E/02, 1:1000; Abcam, Cambridge, USA).

Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Western Blot, Flow Cytometry, Staining, Incubation

Journal: Cancer Science

Article Title: Human leukocyte antigen-E alleles and expression in patients with serous ovarian cancer

doi: 10.1111/cas.12641

Figure Lengend Snippet: Peptide binding and complex stability were analyzed by flow cytometry. (a) To detect the ability of human leukocyte antigen-E (HLA-E) to bind with peptides, cells were stained with 3D12-APC mAb after incubation with 100 μM B7sp peptides for 16 h at 26°C. For HLA-E/B7sp complex thermal stability assay, cells were incubated with 100 μM B7sp peptides for 16 h at 26°C, then further incubated at 37°C for 2 h with or without 1000 ng/mL Brefeldin A. Antibody 3D12-APC mAb was also used for staining. (b) Results were expressed as mean ± SD for three replicate experiments. ** P < 0.01.

Article Snippet: After boiling in 10 mmol/L of citrate buffer (pH 6.0) for antigen retrieval and cooling down, the sections were blocked with 1% BSA and incubated overnight at 4°C with primary mouse monoclonal anti-HLA-E antibody (MEM-E/02, 1:1000; Abcam, Cambridge, USA).

Techniques: Binding Assay, Flow Cytometry, Staining, Incubation, Stability Assay

Journal: Cancer Science

Article Title: Human leukocyte antigen-E alleles and expression in patients with serous ovarian cancer

doi: 10.1111/cas.12641

Figure Lengend Snippet: Cells transfected with the HLA-E*0103 allele escaped from natural killer (NK) lysis. Target cells were cocultured with NK cells at 37°C after incubation for 16 h at 26°C with B7sp peptides. After another 2 h, the percentage of NK lysis was calculated. Results were expressed as mean ± SD for three replicate experiments. ** P < 0.01 versus K562G-E*0101 + B7sp.

Article Snippet: After boiling in 10 mmol/L of citrate buffer (pH 6.0) for antigen retrieval and cooling down, the sections were blocked with 1% BSA and incubated overnight at 4°C with primary mouse monoclonal anti-HLA-E antibody (MEM-E/02, 1:1000; Abcam, Cambridge, USA).

Techniques: Transfection, Lysis, Incubation

Journal: Cancers

Article Title: Infection of Epstein–Barr Virus in Type III Latency Modulates Biogenesis of Exosomes and the Expression Profile of Exosomal miRNAs in the Burkitt Lymphoma Mutu Cell Lines

doi: 10.3390/cancers10070237

Figure Lengend Snippet: Infection with limited EBV copies is sufficient to promote the biogenesis of exosomes in Mutu III cells. ( A ) Western blot analysis of isolated exosomes. Total cell lysates (TCL; left) and isolated exosomes (right) obtained from Mutu cells were subjected to western blot with antibodies against CD63, Alix, LMP1, Calnexin, GM130, and β-actin; ( B ) Analysis of amounts of protein in isolated exosomes released from Mutu cells. Isolated exosomes were subjected to a Bradford protein assay. Relative amounts of protein are shown. The experiment was performed three times independently and the average and its SD are shown in each condition. N.S., not significant. ** p < 0.01 vs. respective control (Student’s t -test); ( C ) Expression of CD63 and Alix in Mutu cells. The distribution of CD63 (top, green) or Alix (bottom, green) in Mutu − (left), Mutu I (middle) and Mutu III cells (right) was analyzed by immunofluorescence staining. The nuclei (blue) were counterstained with Hoechst 33342. Scale bars: 10 µm; ( D ) Flowcytometric analysis of expression of CD63 and Alix in Mutu cells. Expression of CD63 or Alix in Mutu cells was measured by flowcytometry. The experiment was performed three times independently and the average and its SD are shown in each condition. N.S., not significant. * p < 0.05, ** p < 0.01 vs. respective control (Student’s t -test); ( E ) FISH analysis of EBV genome in Mutu cells. Mutu − (left), Mutu I (middle) and Mutu III cells (right) were subjected to FISH with a specific probe targets EBV genome. Individual EBV genomes are shown in green. The nuclei (blue) were counterstained with Hoechst 33342. Scale bars: 10 µm; ( F ) Measurement of EBV copy numbers in Mutu cells. Four fields containing 10–20 nuclei were selected randomly and numbers of EBV genomes per cell were analyzed. Average and its SD are shown in each condition. ** p < 0.01 vs. respective control (Student’s t -test); ( G ) A real-time PCR analysis of EBV copy numbers in Mutu cells. DNAs were isolated from Mutu − , Mutu I, Mutu III, BJAB, or Namalwa cells. EBV titers were analyzed by quantitative PCR. EBV-encoded BALF5 gene was amplified. As an internal control, the human rhodopsin gene was used. The experiment was performed three times independently and the average and its SD are shown in each condition. ** p <0.01 vs. respective control (Student’s t -test).

Article Snippet: A 5 μL aliquot of the fraction containing exosomes were confirmed by western blot with mouse anti-CD63 monoclonal antibody (clone MEM-250, Abnova, Taipei, Taiwan, 1:1000 dilution), mouse anti-LMP1 monoclonal antibody (clone S12, kindly provided by Dr. Teruhito Yasui, National Institutes of Biomedical Innovation, Health and Nutrition, 1:10,000 dilution), mouse anti-Alix monoclonal antibody (clone 3A9, BioLegend, San Diego, CA, USA, 1:1000 dilution), rabbit anti-calnexin polyclonal antibody (Cell Signaling Technology, Trask Lane, MA, USA, 1:1,000 dilution), and rabbit anti-GM130 monoclonal antibody (clone D6B1, Cell Signaling Technology, 1:1000 dilution).

Techniques: Infection, Western Blot, Isolation, Bradford Protein Assay, Control, Expressing, Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Amplification