Supplemental Figure Legend
S. Figure 1
Dependence of K-Ras mutant cells on K-Ras in monolayer and anchorage independent culture conditions.
(A.) Cells were treated as described in Fig. 1. Data were shown as the mean ± S.D. of quadruplicate samples. Repeated experiments gave similar results. (B) Immunoblot analysis to confirm the effects of siRNA targeting of K-Ras. Western blot analysis was performed 3 days after siRNA transfection. Repeated experiments gave similar results.
S. Figure 2
K-Ras is required for anchorage independent growth in a broad panel of K-Ras mutant cells.
Cells were treated as described in Fig. 1. Data were shown as the mean ± S.D. of quadruplicate samples. Repeated experiments gave similar results.
HGF, but not EGF, plays an essential role in proliferation of cells in anchorage independent culture conditions.
S. Figure 3
HGF, but not EGF, plays an essential role in proliferation of cells in anchorage independent culture conditions.
Cells were cultured in medium supplemented with 10% FBS (A and B) or in serum free medium with B-27 supplement (Life Technologies) (C) on normal cell culture plates (monolayer, left panels) or ultra-low attachment plates (anchorage independent, right panels). Cells were stimulated with the indicated concentration of HGF or EGF with or without 1 µM PHA-665752 (PHA) for 3 days. Data were shown as the mean ± S.D. of quadruplicate samples. Repeated experiments gave similar results.
S. Figure 4
Sensitivity to MEK1/2 inhibitor (GSK1120212) in monolayer and anchorage independent culture conditions.
Capan 1 cells were incubated with various concentrations of GSK1120212 for 72 hours on normal cell culture plates (monolayer) or ultra-low attachment plates (anchorage independent). Data were shown as the mean ± S.D. of quadruplicate samples.
S. Figure 5
Activation of Met signaling rescues K-Ras knock down-mediated growth suppression.
Untransfected A549 cells or A549 cells stably transfected with Met(M1268T) were treated and analyzed as described in Fig. 5.
S Figure 6
Anchorage independent culture conditions enhance Met translation
(A) Capan1 and Suit2 cells were seeded on normal cell culture plates (monolayer) or ultra-low attachment plates (anchorage independent) and incubated for 3 days. Cells were then treated with 10 µg/mL of cycloheximide (CHX) for the indicated times. Cell lysates were immunoblotted with the indicated antibodies. (B) qPCR analysis of Met mRNA levels in polysomal fractions for Suit2 cells under monolayer and anchorage independent culture conditions. (C) Capan1 cells were cultured on normal cell culture plates (monolayer: Mo) or ultra-low attachment plates (anchorage independent: AI) for the indicated times. Relative expression levels of Met, eIF4G3, eIF4G1, eIF4G2 and eIF4E mRNAs were evaluated by qPCR. Data were shown as the mean ± S.D. of triplicate samples. Repeated experiments gave similar results.