Journal: Cell Death & Disease

Article Title: Nerve growth factor from Chinese cobra venom stimulates chondrogenic differentiation of mesenchymal stem cells

doi: 10.1038/cddis.2017.208

Figure Lengend Snippet: Effects of NGF on BMSCs in monolayer cultures. ( a ) The MTT assay was used to detect the cytotoxic effects of NGF on BMSCs. (The values are means±S.D., n =6, * P <0.05). ( b ) The effect of NGF on the viability of BMSCs cultured in monolayers for 21 days. Scale bar, 100 μ m. ( c ) Quantification of the DNA content at 7, 14 and 21 days of culture. ( d ) HE staining was used to determine the morphology of BMCSs cultured in monolayers for 21 days. Scale bar, 100 μ m. ( e ) GAG secretion from the cells after treatment for 7, 14 and 21 days. ( f ) qRT-PCR was used to determine the expression of the ACAN , SOX9 , COL2A1 , COL1A1 , RUNX2 , ENO2, GDNF , BDNF and CNTF genes on days 7, 14 and 21. ( g and h ) Immunohistochemistry. BMSCs were stained with COL1A1 ( g ) and COL2A1 ( h ) antibodies after 21 days in culture. Scale bar, 100 μ m. The values are means±S.D., n =6; bars with different letters are significantly different from each other at P< 0.05

Article Snippet: After 1 : 200 dilutions, mouse anti-rabbit collagen type I (COL1A1, Acris OriGene Technologies, Inc., Rockville, MD, USA, TA342814) and collagen type II (COL2A1, Acris Antibodies GmbH, AF5710) antibodies were added to the cells per ections overnight.

Techniques: MTT Assay, Cell Culture, Staining, Quantitative RT-PCR, Expressing, Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Nerve growth factor from Chinese cobra venom stimulates chondrogenic differentiation of mesenchymal stem cells

doi: 10.1038/cddis.2017.208

Figure Lengend Snippet: Chondrogenic effects of NGF on BMSCs grown in 3D cultures. ( a and b ) Immunohistochemical staining of COL1A1 ( a ) and COL2A1 ( b ) was performed in the cultured constructs on days 7, 14 and 21. ( c ) qRT-PCR was performed to determine the expression levels of ACAN , SOX9 , COL2A1 , COL1A1 , RUNX2, ENO2, GDNF , BDNF and CNTF in the cultured constructs on 7 14 and 21 day. ( d ) Western blots was used to analyze the protein expression levels of Col1A1 and COL2A1. The values are means±S.D., n =6; bars with different letters are significantly different from each other at P< 0.05, Scale bar=100 μ m

Article Snippet: After 1 : 200 dilutions, mouse anti-rabbit collagen type I (COL1A1, Acris OriGene Technologies, Inc., Rockville, MD, USA, TA342814) and collagen type II (COL2A1, Acris Antibodies GmbH, AF5710) antibodies were added to the cells per ections overnight.

Techniques: Immunohistochemical staining, Staining, Cell Culture, Construct, Quantitative RT-PCR, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: Nerve growth factor from Chinese cobra venom stimulates chondrogenic differentiation of mesenchymal stem cells

doi: 10.1038/cddis.2017.208

Figure Lengend Snippet: Chondrogenic effects of NGF on BMSCs in vivo . ( a ) Safranin O staining were performed on cartilage sections. ( b and c ) Immunohistochemical staining of COL1A2 ( b ) and COL1A1 ( c ) were performed in cartilage sections. (Original low magnification × 40 (scale bar, 500 μ m); original high magnification × 200 (scale bar, 100 μ m))

Article Snippet: After 1 : 200 dilutions, mouse anti-rabbit collagen type I (COL1A1, Acris OriGene Technologies, Inc., Rockville, MD, USA, TA342814) and collagen type II (COL2A1, Acris Antibodies GmbH, AF5710) antibodies were added to the cells per ections overnight.

Techniques: In Vivo, Staining, Immunohistochemical staining

Journal: Cell Death & Disease

Article Title: Nerve growth factor from Chinese cobra venom stimulates chondrogenic differentiation of mesenchymal stem cells

doi: 10.1038/cddis.2017.208

Figure Lengend Snippet: The receptors and signaling pathways that are activated in NGF-induced BMSCs in vitro and in vivo . ( a-d ) qRT-PCR was to determine the expression levels of the ACAN , SOX9 , COL2A1 and COL1A1 genes in the repaired model after 4, 8 and 12 weeks. ( e ) Western blots was used to analyze the protein expression levels of Col1A1 and COL2A1. ( f and g ) Western blots was used to analyze the protein expression levels of the NGF receptors TrkA and p75 in vitro ( f ) and in vivo ( g ). ( h and i ) Western blots was used to analyze the protein expression of proteins in the PI3K/AKT signaling pathway, including PI3K, AKT and p-AKT, in vitro ( h ) and in vivo ( i ). ( j and k ) Western blots was used to analyze the expression of proteins in the ERK/MAPK signaling pathway, including ERK, p-ERK, P38 and p-P38, in vitro ( j ) and in vivo ( k ). ( l) Schematic description of the relevant signaling pathways that were activated by NGF. The values are means±S.D., n =10 joints; bars with different letters are significantly different from each other at P< 0.05

Article Snippet: After 1 : 200 dilutions, mouse anti-rabbit collagen type I (COL1A1, Acris OriGene Technologies, Inc., Rockville, MD, USA, TA342814) and collagen type II (COL2A1, Acris Antibodies GmbH, AF5710) antibodies were added to the cells per ections overnight.

Techniques: In Vitro, In Vivo, Quantitative RT-PCR, Expressing, Western Blot

Journal: Cell Death & Disease

Article Title: Nerve growth factor from Chinese cobra venom stimulates chondrogenic differentiation of mesenchymal stem cells

doi: 10.1038/cddis.2017.208

Figure Lengend Snippet: Primer sequences used in qRT-PCR experiments

Article Snippet: After 1 : 200 dilutions, mouse anti-rabbit collagen type I (COL1A1, Acris OriGene Technologies, Inc., Rockville, MD, USA, TA342814) and collagen type II (COL2A1, Acris Antibodies GmbH, AF5710) antibodies were added to the cells per ections overnight.

Techniques:

Journal: Arthritis Research & Therapy

Article Title: Cellular and extracellular matrix changes in anterior cruciate ligaments during human knee aging and osteoarthritis

doi: 10.1186/ar4165

Figure Lengend Snippet: Collagen types I, II, and III . (A-D) Collagen type I, (E-H) collagen type II, (I-L) collagen type III (A, E, I) ACL from young normal knee; (B, F, J) ACL from aging knee; (C, G, K) fibroblast-like cell aggregates in the degenerated ACL; (D, H, L) chondrocyte-like cell aggregates in the degenerated ACL. Most collagen bundles were type I collagen positive (A-D) , however, staining intensity of the ECM around chondrocyte-like cell aggregates was lower (black arrows). Type II collagen-positive area is observed around chondrocyte-like cell aggregates (white arrows). In the normal ACL, type III collagen is located within the loose connective tissue (black arrowheads) that divides the collagen fibrils of the ligament into small bundles but not dense collagenous tissues. In the degenerated ACL, type III collagen (white arrowheads). (Original magnification ×40).

Article Snippet: Sections were incubated overnight at 4°C with primary antibodies against MMP-1 (mab901, R&D Systems, Minneapolis, MN, USA; 2 μg/ml), MMP-3 (sc-6839, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:100 dilution), MMP-13 (MAB3321, Chemicon International, Temecula, CA, USA; 1:1,000 dilution), Sox9 (AB5535, Chemicon International, Temecula, CA, USA; 2 μg/ml), Runx2 (sc-10758, Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:50 dilution), Scleraxis (ab58655, Abcam, Cambridge, MA, USA; 5 μg/ml), Ki-67 (ab15580, Abcam; 1:200 dilution), α-SMA (ab5694, Abcam, Cambridge, MA, USA; 1:300 dilution), STRO-1 (mab1038, R&D Systems, Minneapolis, MN, USA; 0.5 μg/ml), collagen I (ab292, Abcam, Cambridge, MA, USA; 1 μg/ml), collagen II (II-II6B3, Hybridoma Bank, Iowa City, IA; 2 μg/ml), collagen III (AP07843PU-N, Acris Antibodies, San Diego, CA, USA; 2 μg/ml), collagen X (ab49945, Abcam, Cambridge, MA, USA; 1:2,000 dilution), and aggrecan (ab3773, Abcam, Cambridge, MA, USA; 1:40 dilution), or negative controls (normal goat IgG, rabbit IgG, mouse IgG or IgM; 1 μg/ml).

Techniques: Staining