Supplemental Materials

This article contains the following supporting material:

• Movie 1 - (4.1 MB) Time-lapse phase-contrast movie of IKD-F11 Fas+ cells migrating on 15 nM LN-coated glass. See Fig. 2A.
  Images were acquired every 4 min for 132 min with a 40 min/s (10 frames/s) display rate.
• Movie 2 - (4.5 MB) Time-lapse phase-contrast movie of IKD-F11 Fas- cells migrating on 15 nM LN-coated glass. See Fig. 2A.
  Images were acquired every 4 min for 132 min with a 40 min/s (10 frames/s) display rate.
• Movie 3 - (4.9 MB) Fluorescence time-lapse confocal microscopy movie of IKD-F11 Fas+ cells transiently expressing mRFP-paxillin migrating on 15 nM LN-coated glass. Images were acquired every 20 s for 20 min with 10 frames/s display rate. See Figure 3E and 3F.
• Movie 4 - (3.9 MB) Fluorescence time-lapse confocal microscopy movie of IKD-F11 Fas- cells transiently expressing mRFP-paxillin migrating on 15 nM LN-coated glass. Images were acquired every 20 s for 20 min with 10 frames/s display rate. See Figure 3E and 3F.
• Movie 5 - (5.4 MB) Time-lapse phase-contrast movie of IKD-F11 Fas+ cells pre-treated with NSC23766 migrating on 15 nM LN-coated glass. Images were acquired every 4 min for 164 min with a 40 min/s (10 frames/s) display rate.
• Movie 6 - (5.2 MB) Time-lapse phase-contrast movie of IKD-F11 Fas- cells pre-treated with NSC23766 migrating on 15 nM LN-coated glass. Images were acquired every 4 min for 164 min with a 40 min/s (10 frames/s) display rate.
• Movie 7 - (5.2 MB) Time-lapse phase-contrast movie of GFP-XtfascinS33A-rescued IKD-F11 (Fas-) cells migrating on 15 nM LN-coated glass. Images were acquired every 4 min for 172 min with a 40 min/s (10 frames/s) display rate. See Fig. 9C.
• Movie 8 - (5 MB) Time-lapse phase-contrast movie of GFP-XtfascinS33D-rescued IKD-F11 (Fas-) cells migrating on 15 nM LN-coated glass. Images were acquired every 4 min for 172 min with a 40 min/s (10 frames/s) display rate. See Fig. 9C.
• Supplementary Figure 1 - A, localisation of cytoskeletal markers in IKD-F11 Fas+ and Fas- cells on 15 nM LN as established by immunofluorescence stainings and confocal microscopy. α-actinin and vinculin stains are shown as projections from Z stacks (bars = 20 μm) and phosphotyrosine is shown as a section at the base of the cells (bar =10 μm).
  B, C, SW480-Pa, IKD-F11 and IKD-F12 cells were cultured in the absence or presence of doxycycline, adhered to LN for 2 h, and 10 μg of whole cell lysates immunoblotted for phosphotyrosine (B), or the indicated cytoskeletal components (C). Molecular mass markers are in kDa.
• Supplementary Figure 2 - Stable expression of GFP and GFP-Xtfascins in F11 sublines. A, Extracts of the indicated lines, prepared from cells cultured without or with exposure to 0.5 μg/ml doxycycline for 48 h, were probed for GFP (two strips from the same X-ray film are shown) or for fascin, using antibody reactive with both human and X. tropicalis fascin (fascin and GFP-fascin bands arrowed in the top panel). This antibody crossreacts with a non-specific band in SW480 cells (asterisk in top panel). B, relative expression levels of human (shaded bars) and X. tropicalis (white bars) fascins in the absence or presence of doxycycline. Fascin bands from immunoblots were calibrated for protein loading against β-actin by image densitometry and all values normalised to the fascin content of SW480 cells in the absence of doxycycline. The numbers on each column show the normalised mean content of human and Xenopus fascin in each cell line under each condition. Columns represent mean values, bars indicate SEM.
• Supplementary Table 1 - Anchorage-independent growth properties of SW480-Pa and IKD clones in the absence or presence of 0.5 μg/ml doxycycline.