Effect of hypoxia-induced cell surface MT1-MMP on cell surface CD44 and mammosphere formation.

<p>A–B) Cell surface CD44 inversely correlates with hypoxia-induced cell surface MT1-MMP: SK-3rd TICs were cultured in the presence of CoCl₂ (Pre CoCl₂; 250 µM) for 24 hours and then switched to CoCl₂-free medium (Post CoCl₂) for indicated times. Flow cytometry analysis employing APC-anti-CD44 antibodies and anti-MT1-MMP antibodies in conjunction with FITC-labeled secondary antibodies (A). Cell surface proteins were biotinylated and examined for CD44 appearance (B, Upper panel). Shed CD44 in the conditioned medium was examined by Western blotting using anti-CD44 antibodies (B, Middle panel). β-actin was used as a loading control (B, Lower panel). Ponceau staining of the membrane with samples from the conditioned medium was employed as a loading control. C) Effect on cell adhesion by hypoxia using a hyaluronic acid adhesion assay: SK-3rd TICs treated with or without CoCl₂ (250 µM) for 24 hours were examined for their ability to adhere to hyaluronic acid-coated 96-well tissue culture plates. The adherent cells were enumerated based on calcein staining. D) Dissociation of mammospheres by low oxygen (Upper panel) or CoCl₂ treatment: Mammospheres from SK-3rd TICs were cultured under either 21% O₂ or 1% O₂ or treated with CoCl₂ (250 µM) for indicated times. The cells were microscopically examined daily. Bar, 100 µm. E) Effect of MT1-MMP on mammosphere forming ability and cell surface CD44 expression: SK-3rd TICs were infected with retrovirus containing cDNAs for GFP or MT1- GFP and shRNAs against luciferase (shRNA-Luc) or MT1-MMP (shRNA-MT1). The cells were cultured in ultra-low attachment culture dishes for their mammosphere forming abilities (Upper panel) and cell surface expression of CD44 and CD24 were examined by flow cytometry using corresponding antibodies (Lower panel). F) Reversal of hypoxia-induced MT1-MMP under normoxic conditions: SK-3rd TICs were cultured under 1% O₂ for 24 hours and then switched to normoxic conditions for indicated time. Flow cytometry analysis was performed using anti-CD44 and anti-MT1-MMP antibodies. NT refers to non-hypoxia-cultured cells.