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The effect of galectin-1 on the differentiation of fibroblasts and myoblasts in vitro

¹ Department of Neuromuscular Diseases, Division of Neuroscience and Psychological Medicine, Faculty of Medicine, Imperial College of Science, Technology and Medicine, Charing Cross Campus, St Dunstan’s Road, London W6 8RP, UK
² Randall Centre for Molecular Mechanisms of Cell Function, King’s College London, New Hunt’s House, Guy’s Campus, London SE1 1UL, UK

View larger version (58K): [in a new window]   Fig. 1. (A) Western blot developed using an anti-galectin-1 antibody. Lane 1, molecular weight markers; lanes 2 and 4, no samples were run; lane 3, galectin-1 positive control; lane 5, C2C12-conditioned media; lane 6, media from COS-1 cells transfected with the galectin-1 construct; lane 7, the non-transfected COS-1 media. The positive galectin-1 band is at approximately 14.5 kDa in lanes 3, 5 and 6. (B) Dot-blot developed using an anti-galectin-1 antibody. Samples 1, 2 and 3 are purified recombinant galectin-1 (1.85 mg/ml) diluted 1:10, 1:50 and 1:100, respectively. Sample 4 is non-diluted GAL-M.

View larger version (92K): [in a new window]   Fig. 2. Cloned dermal fibroblasts grown in 100:1 FCS-20:GAL-M. All cells in this field are desmin positive (A) indicating 100% conversion to a myogenic lineage. Cloned dermal fibroblasts grown in FCS-20 alone are desmin negative (B). Cloned cells are all positive for the mesenchymal cell marker vimentin both in the presence (C) and absence (D) of galectin-1. Nuclei are stained with DAPI. Bars, 50 µm.

View larger version (52K): [in a new window]   Fig. 3. C2C12 cells grown in FCS-20+GAL-M after 2-3 days of growth. Cells stain positively for the muscle-specific marker desmin. A myotube with three nuclei is evident (A). All cells stain positively for vimentin, a mesenchymal cell marker (B) and terminally differentiated cells are positive for myosin II heavy-chain (C). Nuclei are labelled with DAPI in B and C. Bars, 50 µm.

View larger version (205K): [in a new window]   Fig. 4. C2C12 cells in HS-2 (A); HS-2+GAL-M (B); HS-2+COS-1 (C); FCS-20 (D); FCS-20+GAL-M (E); or FCS-20+COS-1 (F). Giemsa stained cultures showing the presence of mononuclear cells and varying numbers of multinucleate myotubes. Bars, 50 µm.

View larger version (14K): [in a new window]   Fig. 5. Averages from seven separate experiments of C2C12 cells grown in HS-2; HS-2+GAL-M; HS-2+COS-1; FCS-20; FCS-20+GAL-M, or FCS-20+COS-1. Results show percentage of nuclei present in myotubes. Asterisk (*) indicates a significant difference from FCS-20 or FCS-20+COS-1 tested by one-way ANOVA and post-hoc multiple paired comparison (P<0.05). Higher percentages of nuclei in myotubes were observed in galectin-1-treated cultures.

View larger version (21K): [in a new window]   Fig. 6. Proliferation assays were undertaken on seven separate C2C12 cultures grown in FCS-20; FCS-20+GAL-M, or FCS-20+COS-1 (A) and HS-2; HS-2+GAL-M; HS-2+COS-1 (B). No significant differences were observed in proliferation of cells at any of the time points examined between the various media types.

View larger version (52K): [in a new window]   Fig. 7. Primary myoblast cultures grown in FCS-20 for 2-3 days. Myoblasts are positive for desmin, whereas the muscle fibroblasts are negative (A). All cells stain positive for vimentin (B), and myoblasts, but not fibroblasts, are positive for myosin II heavy-chain (C). Nuclei are stained with DAPI. Bars, 50 µm.

View larger version (187K): [in a new window]   Fig. 8. Giemsa stained primary myoblast cultures grown for 5-6 days in HS-2 (A); HS-2+GAL-M (B); HS-2+COS-1 (C); FCS-20 (D); FCS-20+GAL-M (E); or FCS-20+COS-1 (F). Three different cell types, myoblasts, fibroblasts and myotubes, are present in these populations. Fibroblasts (A, white arrow), myotubes (B, white arrow) and myoblasts (C, white arrow) are evident in these cultures. Myotubes in GAL-M-treated cultures appeared more numerous and larger than in other media types (arrows, B,E). Bars, 50 µm.

View larger version (11K): [in a new window]   Fig. 9. Averages from seven separate experiments of primary myoblast cultures grown in HS-2; HS-2+GAL-M; HS-2+COS-1; FCS-20; FCS-20+GAL-M; or FCS-20+COS-1. GAL-M cultures produced visibly higher counts. Double asterisk (**) indicates a significant difference from all other cultures. Asterisk (*) indicates a difference from all other cultures apart from cultures grown in HS-2 alone. Inverted v (^) indicates a significant difference from FCS-20 and FCS-20+COS-1. Tested by one-way ANOVA followed by post-hoc multiple paired comparison (P<0.05).

View larger version (91K): [in a new window]   Fig. 10. Primary muscle fibroblasts at passage 2 (A-C) and passage 4 (D-E) grown in HS-2+Gal-M for 2-3 days. Cells are negative for desmin in both the absence (A) and presence of GAL-M (D). Cells are positive for vimentin (B) and negative for myosin II heavy-chain in the absence (C) and presence (E) of GAL-M. Nuclei are stained with DAPI. Bar, 50 µm.

View larger version (138K): [in a new window]   Fig. 11. Giemsa stained primary muscle fibroblasts grown for 5-6 days in HS-2 (A); HS-2+GAL-M (B); FCS-20 (C); and FCS-20+GAL-M (D). No myotubes are present in any of these culture conditions. Bars, 50 µm.

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