Supplementary Figure 1. Effect of E2F1 expression on cell migration and invasion in vitro. A) Invasive and migratory capacity of SK-Mel-103 and SK-Mel-147 cells expressing shE2F1 or control-shRNA analyzed by Boyden chamber (left) and scratch assay (right). Values of invasion were obtained by counting five fields per membrane. The data are means of three separate experiments. Error bars = upper 95% confidence intervals. Mean invasion of shE2F1 cells was set as 1.0 (SK-Mel-147, P = .009; SK-Mel-103, P = .0008; Mann–Whitney U test, two-sided). The scratch migration assay indicates less wound healing by E2F1-knockdown cells. Bar graphs represent means of three independent experiments with error bars showing upper 95% CIs. Values of Ad.shE2F1-infected melanoma cells were set as 1.0. The white area represents the edges of the denuded zone at the beginning of the experiment (top) and the 40 hr time point (bottom) analyzed by ImageJ software. Cell migration was measured as reduction of the wound area in each photographed field. SK-Mel-103, P = .019; SK-Mel-147, P = .004 (Mann–Whitney U test, two-sided). B) Viability of metastatic cells treated as outlined in (A) was measured by TACS XTT assay at indicated times (upper panel). Each data point represents the mean number of viable cells in triplicate dishes. Error bars = upper 95% confidence intervals. Immunofluorescence staining of actively proliferating cells is shown (green, lower panel). Cells were labeled 48 hr after infection with 10 mM EdU (Invitrogen, Karlsruhe, Germany) and visualized by laser scanning microscopy with Alexa Fluor 488. Phase contrast microscopy (lower left panel). Magnification x63. C) Immunofluorescence staining (upper) and immunoblotting (bottom) of E-cadherin levels in SK-Mel-147 cells 48 hrs after knockdown of E2F1 using mouse monoclonal E-cadherin antibody (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA). Blots were probed with mouse monoclonal actin antibody (dilution 1:4000; Sigma-Aldrich, München, Germany) as a control for loading and transfer.

A

shE2F1control-shRNA

Mock

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Ad.shE2F1

control-shRNA

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shE2F1

B C

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Actin

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SK-Mel-147

SK-Mel-103 SK-Mel-147