JCS010488 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. pERK1/2 and pTyrEGFR BioPlex assay development. The phospho-tyrosine EGFR and phospho-ERK1/2 assays were checked for linearity using two approaches. First, lysate from a stimulated parental sample was loaded in increasing amounts. Second, stimulated lysate was mixed at various ratios with an unstimulated lysate, keeping the amount of total protein constant at 10 μg per well. Varying the fraction of stimulated lysate showed linearity for both the (A) pTyrEGFR and (B) pERK1/2 assays, indicating that the extent of phosphorylation can be quantitatively determined.

• Supplemental Figure S2 -

  Fig. S2. Phospho-tyrosine EGFR dynamics. Phospho-tyrosine EGFR dynamics measured using a quantitative immunoprecipitation bead-based pTyrEGFR BioRad Bio-Plex phosphoprotein detection assay. Cells were spiked with 2 nM exogenous EGF (filled circles, squares and diamonds) or blocked with 10 μg/ml of 225 for 2 hours prior to three PBS washes and serum-free media replacement (empty circles and squares). Error represents one standard deviation from the mean of triplicate lysates.

• Supplemental Figure S3 -

  Fig. S3. Cell migration speed during 30-minute intervals. Average cell speed was calculated at 30-minute time intervals. Parental, ECT and TCT cells are shown under serum-free media conditions. In addition, the parental cells are shown under 0.2 and 2 nM EGF stimulation. Error represents the represents standard error of the mean calculated at each 30-minute interval and n>200 for each condition.

• Supplemental Figure S4 -

  Fig. S4. Effects of long-term EGF stimulation on parental cell migration. Cell migration was measured in a sparse-labeled monolayer high-throughput assay. Parental, ECT and TCT cells were starved for 15 hours in serum-free media before adding fresh serum-free media ∼1 hour before imaging. In addition, parental cells were pre-treated with exogenous EGF (0.2, 2 and 25 nM) in serum-free media for 15 hours before adding fresh EGF-containing media 1 hour prior to experimental setup and imaging. Fluorescence images were acquired at 15-minute intervals for 8 hours with a Cellomics KineticScan and subsequently imported in Imaris for single-cell tracking of the labeled cells. Cell paths were analyzed in Matlab. The average cell migration speed is shown under each condition. Error represents s.e.m. and n>200 cells per condition.

• Supplemental Figure S5 -

  Fig. S5. Phospho-ERK measurements under exogenous stimulation and varying ligand release. ERK phosphorylation was measured from triplicate lysates using a BioPlex assay. Cells were starved for 16 hours in serum-free media prior to stimulation. The parental cells were lysed after a 2-hour incubation with serum-free media or exogenous EGF (0.2 and 2 nM). ECT cells were lysed after 2 hours in fresh serum-free media. In addition, TCT cells were lysed after a 2-hour incubation in serum-free media or increasing concentrations of batimastat (0.12, 0.33, 0.86 and 10 μM Bat). Error represents standard deviation from triplicate lysates.