PLC1 is essential for early events in integrin signalling required for cell motility

JCS02374 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 (Adobe PDF) -

  Fig. S1. Motility profiling of BE cancer cells plated on Matrigel. BE cells were analysed by fluorescent video microscopy (Nikon Eclipse Microscope, Simple PCI software). In this experiment, 10% of the BE cells plated on the Matrigel had been previously labelled with fluorescent tracker dye. Still images from the movie were taken and used to illustrate cell motility on Matrigel. The top panel shows cells grouping together in initial cell clusters. Cells 2 and 3 migrate towards each other, with cells 1 and 4 subsequently moving towards the initial cell group. The bottom panel shows the later motility event of guided motility. Cell 2 slides along and underneath Cell 1, whereas Cells 4 and 5 slide past each other and are joined by cell 6 gliding along the network.

• Supplemental Figure 2 (Adobe PDF) -

  Fig. S2. (A) PLCg1 involvement in cell elongation. Mouse fibroblasts, either with PLCg1 knocked-out (PLCg1^–/–) or with PLCg1 added back (PLCg1⁺), were plated on Matrigel and their elongation and motility analysed by microscopy (Nikon Eclipse microscope, Simple PCI software) after 3 hours when the differences were the most prominent. However, over longer periods, the difference between the PLCg1^–/–and PLCg1⁺ fibroblasts became less obvious, suggesting a delayed response in PLCg1^–/– cells (top panels). Stable fibroblast cell line PLCg1⁺ was transiently transfected with the plasmid encoding GFP and the original PLCg1^–/– fibroblasts were transfected with plasmids encoding GFP, the wild-type GFP-PLCg1 or catalytically inactive PLCg1 (co-transfected with GFP for identification purposes) as indicated in the bottom panel. Representative images are shown and average percentage cell elongation for transfected cell lines, analysed from 20 cell images, are reported with standard deviations: 1, PLCg1⁺+GFP; 2, PLCg1^–/–+ GFP; 3, PLCg1^–/–+PLCg1GFP; 4, PLCg1^–/–+inactive PLCg1/GFP. (B) Western blot analysis of PLCg1 and PLCg2 isoform expression in the PLCg1^–/– and PLCg1⁺ fibroblast cell lines. It has been suggested that the levels of PLCg2 are upregulated compared with normal fibroblasts (see text).

• Supplemental Figure 3 (Adobe PDF) -

  Fig. S3. PLCg1 is less involved in BE cell morphological changes on other matrices. BE cells pre-treated with either U73343(2 mM) or U73122(2 mM) were plated on various surfaces/coatings (used in accordance with the manufacturers’ recommendations) and elongation analysed by microscopy (Nikon Eclipse Microscope, Simple PCI software). (A) Representative images are shown after 8 hours. (B) Individual cell elongation was measured from several recorded images using frame-by-frame analysis over 8 hours and a mean average percentage elongation post-plating calculated and reported with standard deviations (n=20): 1, Matrigel; 2, plastic; 3, collagen I; 4, collagen IV; 5, laminin.

• Supplemental Figure 4 (Adobe PDF) -

  Fig. S4. PLCg1 siRNA inhibits formation of actin protrusions. Serum-starved DU145 cells pre-treated with siRNA were serum stimulated for times shown, fixed and stained for F-actin (Phalloidin). Analysis was by Biorad MRC 1024 confocal microscopy (Laser Sharp software).

• Movie 1 -

  All recordings were of cells plated on Matrigel and were obtained by time-lapse video microscopy with one image taken every 10 minutes (Nikon Eclipse Microscope, Simple PCI software). Images were processed (Simple PCI software and Quick Editor) to run at a rate of eight frames per second.

  Movie 1. HUVEC motility on Matrigel.

• Movie 2 -

  Movie 2. DU145 prostate cancer cell motility on Matrigel.

• Movie 3 -

  Movie 3. BE colorectal cancer cell motility on Matrigel.

• Movie 4 -

  Movie 4. BE colorectal cancer cell motility on Matrigel is inhibited by the PLC inhibitor U73122 (2 mM).

• Movie 5 -

  Movie 5. BE colorectal cancer cell motility processes on Matrigel. BE cells plated on Matrigel after pre-labelling (green tracker dye) 10% of the population to enable easy identification of cell motility events. The remaining unlabelled BE cells (90%) are visible as red-edged cells.

• Movie 6 -

  Movie 6. BE colorectal cancer cell motility processes on Matrigel. From Movie 5, non-labelled cells were edited out by Simple PCI software to enable motility of individually labelled cells (grey scale) to be analysed.