Supplemental Materials

This article contains the following supporting material:

• Supplemental Video 1
• Supplemental Video 2
• Supplemental Video 3
• Supplemental Video 4
• Supplemental Video 5
• Supplemental Figure 1 - Three primer PCR was used to determine the genotype of the mice and E13 mice embryos. With the wild-type locus, WAVE1-1F and WAVE1-2R primers bind and generate a 400bp PCR product. With the Scar1 knock-out locus, primer LTR2-2F can now bind specifically to the gene-trap insertion sequence and generate a 150bp PCR product with WAVE1-2R. No PCR product can now be generated between WAVE1-1F and WAVE1-2R because the elongation time of the PCR is too short.
• Supplemental Figure 2 - Confocal images of Scar1 and Scar2 localisation in E13 primary wild-type and Scar1 null MEFs. Wild-type and Scar1 null cells were stimulated for 5 minutes with PDGF (10ng/ml) and then fixed and stained using TRITC phalloidin (F-actin), anti-Scar1 and anti-Scar2 antibodies. Arrows with tails indicate localisation of Scar1 and Scar2 to dorsal ruffles (A, G and J). Unlike Scar1, Scar2 displayed consistent localisation to lamellipodia (arrowheads in G and J). Scale bar: 20μm.
• Supplemental Figure 3 - Titration of PDGF stimulation of E9 immortalised MEFs. The immortalised cells were stimulated for 2, 5 and 7 minutes with different concentrations of PDGF (A) 2.0ng/ml, (B) 1.0ng/ml and (C) 0.5ng/ml). The percentage of cells producing dorsal ruffles at each time point was determined (error bars: ± standard deviation). Student T-test p-values are indicated on graphs.
• Supplemental Figure 4 - The localisation of endogenous Arp2/3 complex, N-WASP and cortactin in PDGF treated primary MEFs. Wild-type MEFs were PDGF stimulated for 5 minutes, fixed and stained with anti-N-WASP (A and E), anti-Arp2 (B), anti-cortactin (F) and Alexa-350 phalloidin (C and G). Arrows indicate colocalisation of N-WASP with Arp2/3 complex and cortactin in dorsal ruffles. Scale bars: 20μm.
• Supplemental Figure 5 - Wild-type E9 immortalised MEFs were cotransfected with either human WASP-GFP and N-WASP siRNA (A-D) or human Arp2-myc and Arp2 siRNA (E-H) for 48 hours. After over-night serum starvation, the cells were PDGF stimulated for 5 minutes before staining for endogenous Arp2/3 complex (anti-p34) and F-actin (Alexa-350 phalloidin). Arp2-myc transfection was detected by 9E10 anti-myc staining (panel E). Arrows indicate colocalisation of Arp2-myc or WASP-GFP in the dorsal ruffle with F-actin and Arp2/3 complex. Scale bars: 20μm.
• Supplemental Figure 6 - The effects of dorsal ruffle formation on stress fibre retention. Serum starved wild-type, Scar1 null, Scar2 siRNA, Arp2 siRNA and N-WASP siRNA treated immortalised MEFs were PDGF stimulated for 15 minutes, fixed and stained with TRITC phalloidin. Arrows indicate loss of stress fibres within the dorsal ruffles. Arrowheads show no disruption to the stress fibres. Scale bar: 20μm.