(A) Montages of DIC from . Time stamps are hrs:min:sec. Scale bar is 50μm. (B) Migration tracks of the cells indicated by red and yellow arrows in A. (C) Western blot analysis of profilin-1 and -2. (D) Quantitation of migration speed; values are averages of mean speed from at least 30 cells ± SEM from three experiments. (E) MCF10A 3D cultures. Left panels are phase-contrast images (arrows indicate cell invasion) and right panels are 3D reconstruction of confocal z- series. Scale bar is 50μm. (F) Boyden chamber invasion assays; values are averages of mean number of invading cells (normalized over control) from three independent experiments ± SEM. (G) Quantitation of profilin-1 (a) and 2 (b) intracellular concentrations. Values are means from three independent experiments ± Std Dev. (H) Wound-healing assay of SUM159 control, PFN1 KD and PFN2 KD cells; plot shows wound areas (normalized over control) from a representative experiment (of three experiments); and right panels show cells at the edge of the wound highlighted in green. Scale bar is 50μm. See also and .

(A) 3D reconstruction of confocal z-series of 3D cultures. Scale bar is 40μm. (B) Boyden chamber invasion assays; values are averages of mean number of invading cells (normalized over control) from three independent experiments ± SEM. (C) Macroscopic view of representative tumors from mammary fat pads injections. (D) (Top row) H and E staining of sections from control, PFN1 KD and PFN2 KD SUM159 tumors. White arrow indicates local invasion. (Bottom row) Left panel shows box plots of the quantitation of invasive foci in tumors from three experiments; middle panel shows an invasive focus at the sentinel lymph node in PFN2 KD tumor (inset is a magnification of the region indicated by a white arrow); and right panel shows a tumor edge with dissociated invasive cells in a PFN2 KD tumor. Scale bar is 50μm. See also .

(A) Montage from time-lapse movies of cells microinjected with Alexa Fluor® 568-conjugated actin (); colored lines indicate the positions of the leading edge in the corresponding cells. Indicated time is in minutes. Scale bar is 10μm. (B) Electron micrographs of cortical F-actin cytoskeletons. Arrow indicates a small protrusion (green shade) next to an actin bundles (red shade). Insets are higher resolution images. Scale bar is 1μm. (C) Quantitation of cells with prominent stress fibers (~200 cells were analyzed per group) and (D) Representative images; arrow indicate small protrusion and scale bar is 10 μm. See also and .

(A) Montages of DIC images selected from a segment of at one-hour intervals. Scale bar is 20μm. (B) Kymography analysis: (left) DIC images from at time 0; and (right) minimum projections (showing regions of protrusive activity) of entire time series (acquired at a rate of one frame/sec). Lines indicate the position at which kymographs were registered. Scale bar is 20μm. (C) kymographs from the corresponding movies in (B). Vertical scale bar is 20μm. Horizontal scale bar is two minutes. (D) Average retraction and protrusion speeds; and (E) frequency. Values are averages of means from at least 30 cells (pooled from 3 different experiments) ± SEM. (F) Montages of DIC images selected from a segment of at one- hour intervals. Yellow dextran marks the injected cells. Scale bar is 20μm. (G) Kymography analysis: (left) DIC images from at time 0; and (right) minimum projections of entire time series (acquired at a rate of one frame/sec). Labeled dextran (cyan) was co-injected to identify microinjected cells. Lines indicate the position at which kymographs were registered. Scale bar is 20μm. (H) kymographs from the corresponding movies in (E). Vertical scale bar is 10μm. Horizontal scale bar is one minute. (I) Average retraction and protrusion speed; and (J) frequency. Values are averages of means from at least 30 cells (pooled from three different experiments) ± SEM. See also and and .

(A) Phospho-MLC staining (phospho-Ser19). Insets are magnifications of the areas in the white boxes; white lines trace the cell edge. Scale bar is 10μm. (B) Phospho-MLC WB analysis. (C) Speed (a) and frequency (b) of retraction/protrusion in control and PFN2a overexpressing SUM159 cells with or without ROCK inhibition (). Values are averages of means from at least 30 cells (pooled from 3 different experiments) ± SEM. (D) 3D reconstruction of confocal z-series of SUM159 3D cultures. Scale bar is 40μm. See also and .

(A) High magnification micrographs of the cell edge actin of control, PFN1 KD and PFN2 KD SUM159 cells. Scale bar is 500 nm. (B) (left) Analysis of EVL binding to profilin-1 and 2 in SUM159 cells: HA immuno- precipitation, followed by profilin-1 and 2 western blot (WB); HA-GFP was used as negative control, and HA WB shows the expression levels of HA-GFP and HA-EVL; (right) quantitation of the relative levels of profilin-1 and 2 bound to HA-EVL in SUM159 cells. These data are representative of three independent experiments. Values are averages ± SEM (C) Sedimentation equilibrium analytical ultracentrifugation used to determine the solution molecular weight of monomeric EVL in the presence of profilin-1 or 2a. The sedimentation profile of Cy3-mEVL1-235aa (5μM) alone or combined with either profilin-1 or 2a (40μM) was determined by monitoring the absorbance at 527/550 nm. Global fitting of three equilibrium traces (at three different centrifugation speeds: 10, 14, 20K rpm) for each condition was performed (see Methods for more detail). An extinction coefficient of 79,982 M-1cm-1 (Cy3, 527nm) was used to determine the protein concentration as a function of the radial position. A monomer-dimer model was used to determine the molecular weight for a single ideal species (top panels). (D) (left) Image sequence of filaments polymerizing in vitro in the presence of 2 μM actin (10% Alexa488), plus 2 μM profilin-2a. Barbed end growth of actin filaments was visualized using TIRF microscopy. Top row, 0 nM EVL; bottom row, plus 200 nM EVL. Yellow arrowhead tracks the growth of a single actin filament barbed end. Scale bar is 5 μm. (right) Average barbed-end polymerization rates (subunits/seconds) for single actin filaments in the presence of profilin-1 or 2, plus or minus EVL. In the presence of 1uM actin (10% Alexa488) alone and 100 nM EVL, barbed ends elongated at a rate of 31.1 ± 2.9 sub/sec (unpublished data). Values are averages of polymerization rates from at least 30 filaments pooled from 2–3 slides ± SEM. (E) Control or PFN2 KD SUM159 cells expressing GFP-EVL; (a) still images from , scale bar is 50μm; speed (b) and frequency (c) of retraction/protrusion in control or PFN2 KD cells with or without GFP-EVL expression as calculated from corresponding kymographs. Values are averages of means from at least 30 cells (pooled from 3 different experiments) ± SEM. (F) (Right) F-actin staining in control and PFN2 KD cells with or without EVL overexpression. Scale bar is 50μm. (Left) % of EVL overexpressing (GFP positive) cells with prominent stress fibers (G) 3D reconstruction of confocal z-series of 3D cultures of control and PFN2 KD cells with or without EVL overexpression. Scale bar is 50μm. (H) Q-PCR showing decreased EVL expression after long-term selection in control cells and cells expressing the two shRNAs targeting EVL. (I) F-actin staining and quantitation of reduced bundling. Scale bar is 20μm. (J) Representative wound-healing assay. Values are averages ± Std Dev. (K) Kymograph analyses (images are from ; scale bar is 20μm); values are averages of means from at least 30 cells (pooled from 3 different experiments) ± SEM. See also and .

(A) Confocal microscopy (maximum projection images) of 3D cultures of SUM159 cells on day 8 and after 4 days of induction with doxycycline of control and EVL KD cells using two different inducible shRNA targeting EVL (scale bar is 50 μm); large insets are mid-sections of the confocal z-series and small insets are magnification of the region in the box (scale bar is 10 μm). (B) TurboRFP staining of sections from control, non-induced area of EVL KD SUM159 tumors and of induced EVL KD tumors; arrows indicate extratumoral invasive foci; box-and-whisker plot shows quantitation of invasive foci in the corresponding tumors. Scale bar is 50μm. (C) Confocal microscopy (maximum projection images) of control and EVL KD tumors. Scale bar is 10 μm. Insets are magnified areas within the designated boxes; red channels are shown separately to visualize tumor cell morphology; box-and-whisker plot shows quantitation of the number of protrusions per cell. (D) Model representing the generation of actin bundles by profilin-2/EVL-mediated linear actin polymerization and activated myosin: right panel illustrates the process of actin polymerization mediated by profilin-2, which is summarized in three major steps (middle panel): recruitment (1) and loading (2) of profilin-2:actin by interaction with the profilin-2 PLP binding site; followed by addition of one actin monomer (G-actin) to the barbed end (3). Left panel shows the domain structure of EVL. (E) Model representing the correlation between protrusive activity and the level of actin bundling. See also and and .

(A) Box-and-whisker plots showing relative levels of EVL and PFN2 transcript in grade I, II and III; p values are from ANOVA analysis. (B) Logistic regression analysis of the relationship between transcript level and tumor grade. (C) Kaplan-Meier curves representing the probability of survival of breast cancer patients based on relative levels of EVL expression (green: tumors in the lowest quartile; red: tumors in the highest quartile; and black: the interquartile range). Chi square p values evaluate whether there are significant differences between any of the three groups. Cox p values evaluate the association of expression with survival by treating EVL levels as a continuous variable. (D) Quantitation of EVL and profilin-2 protein expression in normal breast tissue, and in grade I, II and III tumors (n is the number of patients per group; and triplicate sections from each patient were analyzed). Values are averages from visual scores (scale: 1–5) ± SEM. (E) Representation of protein expression versus tumor invasion (invasion was assessed in a blinded fashion: Non-inv is non-invasive group and low-inv and high inv are low and high invasion groups, respectively. Right panel shows the distribution of each group in terms of tumor grade. (F) Representative images of normal and tumor breast tissue. Scale bar is 100μm. Insets are magnifications of the boxed areas. (G) Analysis of actin density in normal and tumor breast tissue (actin density was assessed in a blinded fashion, as described in D). Values are averages from visual scores (scale: 1–5) ± SEM. (H) Correlation of EVL levels with actin density (Spearman’s ρ =0.53; p<8×10-6); size of the circles represents the invasive activity in the corresponding tumors. (I) Schematic representation of the correlation between EVL/profilin-2 expression, actin density and invasive behavior, and the potential effect of profilin-2 activity on invasion with or without EVL. See also .