Figure 4.

Interference with Sra-1 and Nap1 expression impairs lamellipodia formation in B16-F1 cells. (A) Time course of protein suppression upon siRNA expression. Samples from mock, Nap1 and Sra-1/PIR121 siRNA-treated B16-F1 cells were taken at days after transfection (tfx) as indicated and analysed for Nap1 or Sra-1 protein levels by Western blotting. Note the virtually complete suppression of Nap1 expression at days 3–5 and the significant reduction of Sra-1 at day 4 after transfection. (B–C′). Panels show the actin organization in representative B16-F1 cell populations treated with mock (B) and Nap1 siRNAs (C), respectively. (B′) and (C′) depict the cells quantified from (B) and (C), respectively. Only those cells that were mostly visible within the respective fields were counted and classified (B′, C′). Note the absence of lamellipodia in (C) as opposed to (B). Scale bar equals 20 μm. (D) Results of lamellipodia quantification. Values are means±standard errors of means from three independent experiments. Cells were classified according to the categories (▪) with or (□) without lamellipodia as well as (▪) with ambiguous morphology. Note the correlation between Nap-1 and Sra-1 suppression (A) and reduction of the percentage of cells with lamellipodia (D). (E) Rac expression and activation by AlF is unchanged upon RNAi-mediated knockdown of Sra-1 and Nap1. Wild-type B16-F1 cells as well as mock, Sra-1 or Nap1 siRNA-treated cells express equal amounts of Rac (E, input). In addition, increased amounts of active Rac can be precipitated with PAK–PBD from all lysates upon AlF stimulation (E, PAK–PBD).