Supplemental Methods: Cell Culture 84A human mammary epithelial cells (HMEC s) were a kind gift from Martha Stampfer (Lawrence Berkeley Laboratory, Berkeley CA). Cells were maintained in DFCI- medium supplemented with 2.5 ng/ml (Peprotech, Inc.).[, 2] 84A HMEC clone were a kind gift from Steve Wiley (Pacific Northwest National Laboratories, Richland WA) and were maintained in DFCI- medium supplemented with 2.5 ng/ml and 5 μg/ml of Geneticin (Gibco, Invitrogen Inc.). Serum-free DFCI- media is defined here as DFCI- without, bovine pituitary extract, fetal bovine serum, and insulin. Statistics A Lilifors-Test was used to test for normality for speed, persistence, and random motility coefficient data. A Kolmogorov-Smirnov test was used to assess p-values at a 95% confidence interval for non-normal speed, persistence, and random motility coefficient data. P-values for wound healing comparisons were generated using two-sample t-test and ANOVA. All statistics were generated in MATLAB (Mathworks, Inc.). Migration Assay HMECs were seeded in 96-well tissue culture plastic plates (Packard View Plate Black, Ref. 65225) at confluence (~5, cells/cm 2 ) and allowed to adhere for 4-6 hours. Media was then removed and cells were serum starved for 2-6 hours. Starved cells were treated for 3 minutes with 9μM 5-chloromethylfluorescein diacetate (CMFDA, Molecular Probes, Inc.) in serum-free media. CMFDA containing media was removed and cells were then treated with new serum-free media, serum-free media containing ( ng/ml), or serum-free media containing -β (8 ng/ml, Sigma). A wound width ~65 μm was scraped in each well and cell movement imaged every 5 minutes for 2-5 hours using Cellomics KineticScan. For individual cell tracking in the monolayer, CMFDA labeled cells were mixed :2 with unlabelled cells and the mixture was then seeded at confluence, serum starved, scraped and imaged as described
above. Kinetics of wound closure were quantified using an in-house analysis software that calculated the wound area at each time point normalized by the initial wound area. A 5-point time averaging algorithm was used to average wound closure in individual wells of equal treatment into a single trajectory at 3 minute intervals. Individual cell trajectories in monolayer were produced using Imaris (Bitplane, Inc.). Only cells that remained in frame throughout the entire experiment were considered. Each fluorescently labeled object was recognized as a single cell in a monolayer using the built-in spots function. Cell tracks of the fluorescent objects over time were generated with a built-in autoregressive motion algorithm [3]. Cell trajectories were then fit to the persistent random walk equation.[4] Supplemental Figure Captions: Figure. Schematic of high-throughput wound healing assay. Fluorescently labeled cells are either diluted with non-labeled cells or directly seeded and grown to confluence in a 96-well plate. Wounds ~65 μm in diameter are scraped in each well and cell movement is then monitored using epi-fluorescent microscopy (Cellomics Kineticscan). The 96-well plate is kept at 37 C and 5% CO 2 throughout the experiment. Movies are then exported and analyzed using MATLAB and Imaris-based software. Monolayer movement is quantified in terms of wound area and individual cell trajectories are defined in Imaris and then further analyzed for cell speed and persistence as described in the main text. Figure 2. Early phase wound closure is similar for all cell treatments. Normalized wound area after.5 hours for and parental cells under ( ng/ml), (8 ng/ml), or serum-free conditions. Average wound area is reported ± SE. Figure 3. Wound closure curves for all wells. Normalized wound area measured every 5 minutes is reported for all wells observed under each condition.
Figure 4. Histogram of raw data: cell speed (μm/hr). Cell speed distributions for and parental cells treated with ( ng.ml), (8 ng/ml), or under serum-free conditions. Total cell number vary from 53-96. Figure 5. Histogram of raw data: cell directional persistence (min). Cell persistence distributions for and parental cells treated with ( ng.ml), (8 ng/ml), or under serum-free conditions. Total cell number vary from 53-96. Figure 6. Histogram of raw data: cell random motility coefficient (μm 2 /hr). Cell random motility coefficient distributions for and parental cells treated with ( ng.ml), (8 ng/ml), or under serum-free conditions. Total cell number vary from 53-96.. Band, V. and R. Sager, Distinctive traits of normal and tumor-derived human mammary epithelial cells expressed in a medium that supports long-term growth of both cell types. Proc Natl Acad Sci U S A, 989. 86(4): p. 249-53. 2. Hendriks, B.S., et al., Coregulation of epidermal growth factor receptor/human epidermal growth factor receptor 2 (HER2) levels and locations: quantitative analysis of HER2 overexpression effects. Cancer Res, 23. 63(5): p. 3-7. 3. Veenman, C., M. Reinders, and E. Backer, Resolving motion correspondence for densely moving points. IEEE TRANSACTIONS ON PATTERN ANALYSIS AND MACHINE INTELLIGENCE, 2. 23(): p. 54-72. 4. Dickinson, R. and R. Tranquillo, Optimal estimation of cell-movement indexes from the statistical-analysis of cell tracking data. AICHE Journal, 993. 39(2): p. 995-2.
Supplemental Figure { { 96-well plate. Cells are flourescently labelled.. Flourescently labelled cells 2. Mechanical scrape produces mixed with unlabelled cells. ~65 μm wound. 2. Mechanical scrape produces 3. Wound area quantified every ~65 μm wound. 5 minutes. 3. Flourescent cell speed and persistence quantified from cell tracks. 37 C and 5% CO2 Metric: Normalized Wound Area as a function of Time TIME Metric: Cell Speed and Persistence Cell Trajectories as a function of Time
Supplemental Figure 2 SF SF
Supplemental Figure 3 A. Serum Free B..2.2 Serum Free.8.8.6.6.2.2 2 4 6 8 2 4 6 2 4 6 8 2 4 6 Normalized Wound Area C. D..2.2.8.8.6.6.2.2 2 4 6 8 2 4 6 2 4 6 8 2 4 6 E. F..2.2.8.8.6.6.2.2 2 4 6 8 2 4 6 2 4 6 8 2 4 6 Time (hours)
Supplemental Figure 4.5.5 5 5 Serum Free.35.3.25.2.5..35.3.25.2.5..5.5 2 3 4 5 6 7 8 9.5 5.35.3.25.2.5..5 2 3 4 5 6 7 8 9.5 2 3 4 5 6 7 8 9.5 5.35.3.25.2.5..5 2 3 4 5 6 7 8 9.5 5 5.35.3.25.2.5..5.35.3.25.2.5..5 2 3 4 5 6 7 8 9 Speed (μm/hr) 2 3 4 5 6 7 8 9
Supplemental Figure 5 Serum Free.5 5.35.3.25.2.5..5.5 5.35.3.25.2.5..5 5 5 2 25 3 35.5 5.35.3.25.2.5..5 5 5 2 25 3 35.5 5.35.3.25.2.5..5 5 5 2 25 3 35 5 5 2 25 3 35.5 5.35.3.25.2.5..5 5 5 2 25 3 35.5 5.35.3.25.2.5..5 Persistence (min) 5 5 2 25 3 35
Supplemental Figure 6 Serum Free.9.8.7.6.5.3.2..9.8.7.6.5.3.2. 2 3 4 5 6 7 8 9.9.8.7.6.5.3.2. 2 3 4 5 6 7 8 9.9.8.7.6.5.3.2. 2 3 4 5 6 7 8 9 Random Motility Coefficient (μm 2 /hr) 2 3 4 5 6 7 8 9.9.8.7.6.5.3.2. 2 3 4 5 6 7 8 9.9.8.7.6.5.3.2. 2 3 4 5 6 7 8 9