Journal: The Journal of Cell Biology

Article Title: Regulated Production of Mineralization-competent Matrix Vesicles in Hypertrophic Chondrocytes

doi:

Figure Lengend Snippet: Proteoglycan content in control and mineralizing matrix vesicle preparations. Aliquots of matrix vesicle preparations isolated from control cultures ( B , Control ) or vitamin C/phosphate-treated chondrocyte cultures ( B , Vit.C/P i ) were dotted onto nitrocellulose filters and immunostained with antibodies against aggrecan as described in Materials and Methods. ( A ) Different concentrations of purified aggrecan were dotted onto nitrocellulose filters and immunostained with antibodies against aggrecan. Densitometry was used to calculate the amount of aggrecan in the vesicle preparations using the staining intensities of the aggrecan dots as a standard curve. ( C and D ) EDS analysis of control matrix vesicles ( C ) and mineralizing matrix vesicles ( D ). Note that both vesicle preparations contain similar amounts of sulfur (peaks labeled with S ; 77 counts in control matrix vesicles; 64 counts in mineralizing matrix vesicles). Also note that Ca 2+ peaks are only present in mineralizing matrix vesicles ( D , peaks labeled with Ca ); in addition, mineralizing matrix vesicles contain more phosphate (peaks labeled with P ) than control matrix vesicles.

Article Snippet: After blocking with low fat milk powder the filters were immunostained with polyclonal antibodies against chicken aggrecan followed by secondary horseradish-conjugated anti–rabbit IgG (Rockland Inc., Gilbertsville, PA) and α-chloronaphthol as color substrate.

Techniques: Isolation, Purification, Staining, Labeling

Journal: PLoS ONE

Article Title: Upregulation of glycosaminoglycan synthesis by Neurotropin in nucleus pulposus cells via stimulation of chondroitin sulfate N-acetylgalactosaminyltransferase 1: A new approach to attenuation of intervertebral disc degeneration

doi: 10.1371/journal.pone.0202640

Figure Lengend Snippet: A) NP cells seeded in 96-well plates were cultured in 10% FBS α-MEM supplemented with 0, 0.1, 0.5, or 1.0 mNU/ml NTP with 50 μg/ml L-ascorbic acid-2-phosphate (AsAP). Cell proliferation was evaluated using the MTT assay on days 3, 5, and 7 after treatment, and values are shown as absorbance at 562 nm. Cell proliferation was enhanced on days 5 and 7 by either addition of AsAP or cotreatment with AsAP and NTP, compared with untreated cells on day 3. Data are presented as the average ± standard error (** p < 0.01, N = 3). B) Immunofluorescence staining of aggrecan in NTP-treated NP cells. Cells were seeded at a density of 3x10 3 cells /cm 2 on glass chamber slides and cultured with NTP. After two weeks of culture, expression of aggrecan core protein increased in the NTP-treated cultures compared with untreated cultures or those treated only with AsAP. Representative images are shown. Green indicates aggrecan, and nuclei are shown in blue by staining with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). NTP concentrations of 0.1 and 1.0 mNU/ml were used. Bars are 50 μm.

Article Snippet: Rabbit polyclonal anti-aggrecan antibody was obtained from Merck (MAB2015, Burlington, MA).

Techniques: Cell Culture, MTT Assay, Immunofluorescence, Staining, Expressing

Journal: PLoS ONE

Article Title: Upregulation of glycosaminoglycan synthesis by Neurotropin in nucleus pulposus cells via stimulation of chondroitin sulfate N-acetylgalactosaminyltransferase 1: A new approach to attenuation of intervertebral disc degeneration

doi: 10.1371/journal.pone.0202640

Figure Lengend Snippet: A) After two weeks of culture, glycosaminoglycan (GAG) content per plate increased in cultures with added AsAP, with or without the addition of NTP. B) DNA content increased in cultures with added AsAP, with or without the addition of NTP. C) GAG content normalized to DNA content was significantly upregulated by addition of 0.1 mNU/ml NTP with AsAP. D) After one week of culture, the effect of NTP on aggrecan ( ACAN ) mRNA expression was measured by real-time quantitative PCR. Gene expression of ACAN normalized to GAPDH showed a tendency to increase with increasing concentrations of NTP. Data are presented as the average ± standard error (** p < 0.01, * p < 0.05 when compared with control, ## p < 0.01, # p < 0.05 when compared with AsAP alone, and †† p < 0.01, when compared between NTP–treated samples, N = 5).

Article Snippet: Rabbit polyclonal anti-aggrecan antibody was obtained from Merck (MAB2015, Burlington, MA).

Techniques: Expressing, Real-time Polymerase Chain Reaction