Journal: Journal of the American College of Cardiology

Article Title: Thrombopoietin contributes to enhanced platelet activation in patients with unstable angina.

doi: 10.1016/j.jacc.2006.04.106

Figure Lengend Snippet: Figure 2. (A) Representative fluorescence-activated cell sorting (FACS) analysis showing the percentage of c-Mpl expression on platelet surface detected in vivo in healthy control subjects (C) and in patients with stable angina (SA) and unstable angina (UA). The solid curve represents fluorescence due to binding of non-specific antimouse immunoglobulin G monoclonal antibody; the open curve represents fluorescence due to binding of anti–c-Mpl monoclonal antibody. (B) Quantification of c-Mpl expression on platelet surface detected in vivo in healthy controls and in patients with SA and UA by FACS analysis. One-way analysis of variance with Newman-Keuls multicomparison test was performed. (C) Representative Western blot analysis of the phosphorylation state of c-Mpl in platelet lysates from healthy control subjects and from patients with SA and UA. Platelets from control subjects or patients were either unstimulated or stimulated with saturating concentrations of recombinant human thrombopoietin (rhTPO). The c-Mpl was phosphorylated in platelets from SA patients and healthy control subjects but not in platelets from UA patients. Shown is a representative experiment out of 4 with similar results.

Article Snippet: For in vitro experiments, 100 l diluted blood from ealthy adult donors was preincubated at 37°C with 25 l lasma from patients or control subjects or recombinant uman TPO (rhTPO) (R&D Systems) for 5 min and then timulated with adenosine 5=-diphosphate (ADP) (0.8 mol/l; Helena Laboratories, Beaumont, Texas) or epiephrine (EPI) (3 mol/l; Helena Laboratories).

Techniques: FACS, Expressing, In Vivo, Control, Binding Assay, Western Blot, Phospho-proteomics, Recombinant

Journal: Theranostics

Article Title: Progerin accumulation in nucleus pulposus cells impairs mitochondrial function and induces intervertebral disc degeneration and therapeutic effects of sulforaphane.

doi: 10.7150/thno.30658

Figure Lengend Snippet: Figure 3. Progerin induces aging-related defects and mitochondrial dysfunction in NP cells. (A) Representative IF images and quantification of LAP2, γH2AX, H3K27me3, and lamin B1 expression in the vector con and progerin groups; n = 5; **P < 0.01. (B) TUNEL staining of NP cells in the vector con and progerin groups. Nuclei were stained with DAPI. The number of TUNEL-positive cells was significantly greater in the progerin group than in the vector con group; n = 5; **P < 0.01. (C) Representative flow cytometry dot plots of apoptosis after Annexin V-FITC/PI dual staining. The relative number of apoptotic cells was greater in the progerin group compared to the vector con group; n = 5; **P < 0.01. (D) Representative images and quantification of DCFDA-based ROS levels in the vector con and progerin groups; n = 5; **P < 0.01. (E) JC-1 staining. The red: green fluorescence ratio reflects changes in the mitochondrial membrane potential of NP cells in the vector con and progerin groups; n = 5; **P < 0.01. (F) ATP production in the vector con and progerin groups; n = 5; *P < 0.05. (G) Relative activities of mitochondrial complex enzymes in NP cells in the vector con and progerin groups; n = 4; *P < 0.05, **P < 0.01. Data represent mean ± SEM. LAP2, lamina-associated polypeptide2; γH2AX, serine-139 phosphorylated H2AX; H3K27me3, heterochromatin-associated tri-methylated lysine 27 on histone 3; SA-β-gal, senescence-associated β-galactosidase; DCFDA, 2',7'-dichloro-dihydrofluorescein-diacetate; JC-1, 5,5-,6,6-tetrachloro-1,1-,3,3-tetraethylbenzimidazole-carbocyanine iodide.

Article Snippet: For immunofluorescence and immunohistochemical analyses, the following antibodies were used: Anti-aggrecan (rabbit polyclonal; Proteintech, #13880-1-AP), P16INK4a mouse monoclonal (Abcam, #ab54210), LAP2 rabbit polyclonal (Proteintech, #14651-1-AP), γH2AX (Proteintech, #10856-1-AP), H3K27me3 rabbit monoclonal (CST, #9733), lamin B1 rabbit polyclonal (Proteintech, #12987-1-AP), progerin mouse monoclonal (Santa Cruz, #sc-81611), and collagen II rabbit polyclonal (Abcam, #ab34712).

Techniques: Expressing, Plasmid Preparation, TUNEL Assay, Staining, Cytometry, Membrane, Methylation

Journal: Theranostics

Article Title: Progerin accumulation in nucleus pulposus cells impairs mitochondrial function and induces intervertebral disc degeneration and therapeutic effects of sulforaphane.

doi: 10.7150/thno.30658

Figure Lengend Snippet: Figure 5. SFN ameliorates progerin-induced aging defects and mitochondrial dysfunction. (A) IF of LAP2, γH2AX, H3K27me3, and lamin B1 in the progerin+vehicle and progerin+SFN groups; n = 5; *P <0.05. (B) Representative images and quantification of ROS levels in the progerin+vehicle and progerin+SFN groups; n = 5; *P < 0.05. (C) JC-1 staining. The red: green fluorescence ratio reflects changes in the mitochondrial membrane potential of NP cells in the progerin+vehicle and progerin+SFN groups; n = 5; *P < 0.05. (D) ATP production in the progerin+vehicle and progerin+SFN groups; n = 5; *P < 0.05. (E) Western blotting analysis of OPA1, Drp1, Mfn1, and Mfn2 in NP cells from the vector+vehicle, progerin+vehicle, and progerin+SFN groups; n = 5; *P < 0.05, **P < 0.01. (F) Western blotting analysis of PGC1α, pAMPK, and AMPK in NP cells from the vector+vehicle, progerin+vehicle, and progerin+SFN groups; n = 5. β-actin was used as the control. *P < 0.05, **P < 0.01. Data represent mean ± SEM. SA-β-gal, senescence-associated β-galactosidase; DCFDA, 2',7'-dichloro-dihydrofluorescein-diacetate; JC-1, 5,5-,6,6-tetrachloro-1,1-,3,3-tetraethylbenzimidazole-carbocyanine iodide; OPA1, optic atrophy-1; Drp1, dynamin-related peptide1; Mfn1, mitofusin1; Mfn2, mitofusin2; PGC1α, peroxisome proliferator-activated receptor-γ coactivator1α; AMPK, AMP-activated protein kinase.

Article Snippet: For immunofluorescence and immunohistochemical analyses, the following antibodies were used: Anti-aggrecan (rabbit polyclonal; Proteintech, #13880-1-AP), P16INK4a mouse monoclonal (Abcam, #ab54210), LAP2 rabbit polyclonal (Proteintech, #14651-1-AP), γH2AX (Proteintech, #10856-1-AP), H3K27me3 rabbit monoclonal (CST, #9733), lamin B1 rabbit polyclonal (Proteintech, #12987-1-AP), progerin mouse monoclonal (Santa Cruz, #sc-81611), and collagen II rabbit polyclonal (Abcam, #ab34712).

Techniques: Staining, Membrane, Western Blot, Plasmid Preparation, Control