g8 mouse fetal myoblasts  (ATCC)

Journal:

Article Title: Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-? and promotes tumor invasion and angiogenesis

doi:

Figure Lengend Snippet: Characterization of the TA3 transfectants and G8 myoblast invasion assays. (A) Schematic representation of v5-tagged MMP-9 and the CD44–MMP-9 fusion protein. Amino acid sequences that form the fusion are indicated. (L) Leader peptide; (pro) prodomain; (Zn) zinc-binding domain (active site); (hemopex) hemopexin domain; (tm) transmembrane domain; (ic) intracellular domain; (v5) v5 peptide tag. (B) (a) Western blot of TA3 transfectant cell lysates and corresponding concentrated serum-free conditioned media (b), with anti-CD44 mAb IM7.8. Endogenous CD44 expression is comparable in all transfectants (a). Soluble CD44 expression is comparable in TA3sCD44, TA3sCD44mmp-9/CD44fp, TA3sCD44mmp-9v5 cells (b, lanes 3–8), whereas TA3wt cells do not express soluble CD44 (lanes 1,2). (c,d) Gelatin zymograms of (c) serum-free media and (d) crude membrane preparations of the transfected TA3 cells. Cell lysates (a), conditioned media (b,c), and crude membrane preparations (d) were from the following: (lanes 1, 2) TA3wt cells; (lanes 3,4) TA3sCD44 cells; (lanes 5,6) TA3sCD44mmp-9/CD44fp cells; (lanes 7,8) TA3sCD44MMP-9v5 cells. Molecular mass markers are indicated. (C) G8 myoblast monolayer invasion assay: Expression of the MMP-9–CD44 fusion protein by TA3sCD44mmp-9/CD44fp cells (a, arrowhead) rescues the ability of TA3sCD44 (b, arrow) to invade the G8 monolayer to an extent that is comparable with that of TA3wt cells (c, arrowhead), whereas overexpression of soluble MMP-9v5 has little effect (d, arrow). Bar, 80 μm.

Article Snippet: Cells and antibodies G8 mouse fetal myoblasts and KM201 and IM7.8 hybridomas, which produce rat-anti-mouse CD44 mAb, were obtained from the American Type Culture Collection (ATCC, Rockville, MD), and cultured in DMEM with 10% FBS (Irvine Scientific, Santa Ana, CA).

Techniques: Binding Assay, Western Blot, Transfection, Expressing, Membrane, Invasion Assay, Over Expression

Journal:

Article Title: Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-? and promotes tumor invasion and angiogenesis

doi:

Figure Lengend Snippet: Endothelial cell tubule formation assay. BME cells (3 × 105/ml) were seeded onto type I collagen gels in medium supplemented with 10% calf serum. On the following day, the medium was aspirated and replaced with conditioned serum-free medium derived from cocultures of G8 myoblast monolayers and TA3wt (A) TA3sCD44 (B), TA3sCD44/MMP-9-CD44fp (C), or TA3sCD44/MMP-9v5 (D) cells. A total of 30 μg/ml of pan specific anti-TGF-β (E) or anti-bFGF (F) antibody were added to the TA3wt/G8-conditioned coculture medium prior to use in the assay. Tubules are indicated by arrows. Bar, 140 μm.

Article Snippet: Cells and antibodies G8 mouse fetal myoblasts and KM201 and IM7.8 hybridomas, which produce rat-anti-mouse CD44 mAb, were obtained from the American Type Culture Collection (ATCC, Rockville, MD), and cultured in DMEM with 10% FBS (Irvine Scientific, Santa Ana, CA).

Techniques: Tube Formation Assay, Derivative Assay

Journal:

Article Title: Cell surface-localized matrix metalloproteinase-9 proteolytically activates TGF-? and promotes tumor invasion and angiogenesis

doi:

Figure Lengend Snippet: CD44-anchored MMP-9 activates TGF-β. (A) The ability of TA3 transfectant-G8 myoblast coculture media to stimulate TMLC luciferase activity is shown. Conditioned coculture media tested are indicated. (B) Pan-specific TGF-β antibody (30 μg/ml) or specific antibodies against TGF-β1 (100 ng/ml), TGF-β2(100ng/ml), or TGF-β3(100 ng/ml) were used to determine the activity of TGF-β isoforms in the coculture media. One unit of luciferase activity corresponds to the activity produced by 5 pg of purified human TGF-β1 (R & D).

Article Snippet: Cells and antibodies G8 mouse fetal myoblasts and KM201 and IM7.8 hybridomas, which produce rat-anti-mouse CD44 mAb, were obtained from the American Type Culture Collection (ATCC, Rockville, MD), and cultured in DMEM with 10% FBS (Irvine Scientific, Santa Ana, CA).

Techniques: Transfection, Luciferase, Activity Assay, Produced, Purification