SLK knockdown or expression of a dominant negative SLK inhibits cell migration.

<p>Subconfluent MEF 3T3 cells were infected with Adenovirus vectors expressing DN SLK (Ad-HA-KΔC) or LacZ control and subjected to fibronectin (FN) transwell migration assays. (A) Western blot analysis of HA-KΔC expression. Cdc42 was used as a loading control. (B) Polycarbonate membranes were DAPI stained and cells on the underside were enumerated (C) in random fields and expressed as the average/field from triplicate wells. (D) MEF 3T3 cells were transfected with SLK siRNAs and analysed for SLK expression. Western blot analysis of treated lysates indicates that SLK siRNA at 10 pM resulted in a marked knockdown of SLK. Reprobing the membrane with a α-tubulin antibody was used as a control for loading (lower panel). (E–F) Cells were treated with SLK-specific or control siRNAs and assayed for migration through a chamber coated with bovine serum albumin (BSA) (10 µg/ml) (control) or fibronectin (FN) (10 µg/ml). In both cases a 60–70% reduction in migration was observed. (G) Confluent MEF3T3 cells were infected with Adenovirus vectors expressing a scramble or SLK shRNA and manually scratched with a pipet tip. Wound closure was followed for 12 h and the percent closure was evaluated.</p

SLK activation requires FAK/src/MAPK signaling.

<p>(A) Confluent MEF 3T3 monolayers were pre-incubated (60 min) with inhibitors and then scratch wounded in the presence of inhibitors. Cells were collected 60 minutes later and analysed for SLK kinase activity. (A) Treatment with PP2 or PP3 control. (B) FAK-null or wildtype cells were subjected to scratch wound assays as above and assayed for SLK activity. (C) Treatment with U0126 and DMSO control. Phospho-Erk1/2 is shown as a control for U0126 treatment. SLK activation requires FAK/src/MAPK signaling.</p

SLK knockdown results in adhesion stabilization.

<p>Monolayers of MEF3T3 on FN were infected with adenovirus expressing shSLK or an shScramble control and scratch wounded. After 2 hours, the cells were fixed and stained for SLK (A, C, E and G) in combination with vinculin (B and D) or phalloidin (F and H). In addition to reduced SLK staining, shSLK expressing cells showed no SLK immunoreactivity at the leading edge with an increased number of focal adhesions. No overt differences were observed in phalloidin stained samples. Scale bar 10μ.</p

Recruitment of SLK at the leading is c-src-dependent.

<p>Confluent monolayers of FAK wildtype (A–B), FAK-null (C–D), SYF +c-src (E–F) and SYF (G–H) cells were scratch wounded and immunostained for SLK and Rac1. Similarly, MEF3T3 monolayers were pretreated with U0126 (30 min), scratch wounded and stained for SLK and Rac1 (I and J). SLK and Rac1 failed to be recruited to the leading edge in SYF cells. Scale bar 10μ</p

SLK is recruited to the leading edge.

<p>MEF 3T3 monolayers on fibronectin-coated coverslips were scratch wounded and allowed to migrate for 2–3 hours. Monolayers were immunostained for SLK in combination with actin (A–C), paxillin (D–F), α-tubulin (G–I), or Rac1 (J–L). In addition to perinuclear staining, SLK was found to be recruited into membrane ruffles (arrowheads) at the leading edge with the other markers surveyed. SLK was not found in mature adhesion complexes as shown by the lack of co-localization between SLK and paxillin in these structures (arrows). All photomicrographs are shown at 400×. Scale bar 10μ.</p

SLK is activated by scratch wounding and is required for cell migration.

<p>SLK is activated by scratch wounding and is required for cell migration.</p