c-Met induces tumor-initiating characteristics through CD44s. (A) 5x104 MHCC97-H cells were plated onto 6-well low adherent culture plates in triplicates. Tumorspheres were collected by centrifugation after 2 weeks . For monolayer cultured cells, 5x104 MHCC97-H were plated on 10 cm plate and cultured for 2 weeks. Media was changed every 2–3 days. MHCC97-H cells were plated in low-adherent cell or monolayer cell culture dishes for two weeks and immunoblotting analysis was confirmed on tumorsphere lysates. The data are representative of three independent experiments. (B) 5x103 MHCC97-H scrambled, c-Met shRNA and CD44s shRNA cells were grown in 6-well low-adherent culture plates for two weeks and the numbers of tumorspheres were counted (40X magnification). The data are representative of two independent experiments and are shown as the mean ± SEM of triplicate plates. (C) CD44s recovers tumorsphere formation. MHCC97-H cells were transfected with c-Met siRNA (25pM) for 24 hrs followed by overexpression of CD44s or pBabe empty vector retrovirus for an additional 48 h (immunoblot) or two weeks (tumorsphere assay). (D) Immunoblot data are representative of two independent experiments. The data for the tumorsphere assay data are representative of two independent experiments and are shown as the mean ± SEM of triplicate wells (40X magnification).