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. Author manuscript; available in PMC: 2016 Jul 15.
Published in final edited form as: Cancer Res. 2015 May 14;75(14):2851–2862. doi: 10.1158/0008-5472.CAN-14-1623

Figure 3. Involvement of Met-driven signaling pathways in the growth of cells in anchorage independent culture conditions.

Figure 3

(A-B) Capan1 (A) or A549 (B) cells were transfected with control siRNA, K-Ras siRNA, or Met siRNAs. 24 h after transfection, cells were harvested and seeded onto normal cell culture plates (monolayer) or ultra-low attachment plates (anchorage independent), and relative cell growth was analyzed 3 days (Capan1) or 4 days (A549) after cell seeding. Data were normalized with each control siRNA transfected sample and shown as the mean ± S.D. of quadruplicate samples. NS; not significant, *; p < 0.05, **; p < 0.01, ***; p < 0.0001 (C) The effects of each siRNA were confirmed by Western blot analysis 3 days after siRNA transfection. (D) Capan1 cells were incubated with medium containing vehicle (DMSO), 500 nM PHA-665752 (PHA) or 500 nM XL184 (XL) in monolayer culture conditions for 2 days. Cell lysates were immunoblotted with the indicated antibodies. Repeated experiments gave similar results. (E) Capan1 cells were seeded on normal cell culture plates (monolayer) or ultra-low attachment plates (anchorage independent) and incubated with the indicated concentrations of PHA-665752 (left panel) or XL184 (right panel) for 4 days. Relative viable cell numbers were determined by quantitating ATP present in each well. Data were shown as the mean ± S.D. of triplicate samples. Repeated experiments gave similar results. (F) Capan1 cells were cultured either in monolayer or anchorage independent culture conditions for 3 days, then the cells were treated with 10 ng/mL of HGF. At the indicated time points, cells were collected and immunoblotted with the indicated antibodies.

(G) Capan1 cells were plated on normal cell culture plates (monolayer) or ultra-low attachment plates (anchorage independent) and cultured for 72 h in the medium containing 0, 10 or 50 ng/mL of HGF. Relative viable cell numbers were determined by quantitating ATP present in each well. Data were shown as the mean ± S.D. of quadruplicate samples.