Effects of transcription factor silencing on transformed phenotypes expressed in KRAS‐transformed cells support hierarchical regulation in the gene‐regulatory.

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Effects of transcription factor silencing on transformed phenotypes expressed in KRAS‐transformed cells support hierarchical regulation in the gene‐regulatory network. Effects of transcription factor silencing on transformed phenotypes expressed in KRAS‐transformed cells support hierarchical regulation in the gene‐regulatory network. (A) Anchorage‐independent proliferation (‘3‐dimensional’ (3D) growth in soft agar cultures). (B) Anchorage‐dependent proliferation (‘2‐dimensional’ (2D) growth in adherent monolayer cultures) of RAS‐ROSE cells 48 h after treatment with scrambled siRNA‐duplex (Sc), transfection reagents only (Mock) and two independent siRNA duplexes targeting each transcription factor as determined by colorimetric XTT assay. Each bar indicates the alteration of cell growth relative to untransfected RAS‐ROSE cells. Upstream transcription factors: Otx1, Gfi1, RelA and downstream transcription factors: Fosl1, Hmga2, Klf6 and JunB (see text). (C) Cell‐cycle analysis of RAS‐ROSE cells 48 h after treatment with scrambled siRNA‐duplex (Sc), transfection reagents only (Mock) and two independent siRNA duplexes targeting each transcription factor. Distribution of cell‐cycle phases G0/G1, S and G2/M was determined by FACS analysis after DNA staining of cells with propidium iodide (Supplementary Figure 8). Each bar shows the number of cells in G0/G1 phase relative to untransfected RAS‐ROSE cells. (D) Representative images of scratch assays to determine migratory activity of RAS‐ROSE cells following transcription factor silencing. Cells were transfected with scrambled siRNA, transfection reagents only (mock), and two independent siRNA duplexes targeting RelA. Pictures were taken before scratching the wound, at time 0 and 19, 24 and 36 h post scratch. Phase contrast, magnification 100‐fold. Migration was strongly inhibited after RelA knockdown as compared with controls. (E) Quantitative analysis of wound closure by RAS‐ROSE cells following transcription factor knockdown as compared with control cells. Bar plots show average and standard deviation of two biological and three technical replicates for two different transfected siRNAs normalized to scrambled control. Source data is available for this figure in the Supplementary Information. Iwona Stelniec‐Klotz et al. Mol Syst Biol 2012;8:601 © as stated in the article, figure or figure legend