Figure 3:

Comparing cellular morphology and invasive behaviors in HCR cell lines. A, Representative images of HCR-WT, HCR-16, and HCR-95 cells plated for 24 hours on gelatin-coated coverslips, stained with phalloidin (grey) and DAPI (pseudocolor: HCR-WT– cyan, HCR-16 – yellow, HCR-95 – magenta). B, Quantification of cell area measurements on phalloidin-stained images (n= 81-82cells/condition). C, Quantification of total F-actin fiber length per cell on phalloidin-stained images (n=75 cells/condition). D, Example of invadopodia structures overlying areas of fluorescent matrix degradation in HCR-95 cells (matrix – grey, degradation puncta - black, cortactin – green, DAPI – magenta pseudocolor). Arrowheads indicate active invadopodia. E, Representative images of each cell line stained with cortactin (grey), F-actin (not shown to enable better visualization of puncta only) and DAPI (pseudocolor: HCR-WT– cyan, HCR-16 – yellow, HCR-95 – magenta) to identify F-actin+/ cortactin+ puncta indicating individual invadopodia structures (arrowheads) and rosette invadopodia superstructure in HCR-95 (arrow with tail). F, Quantification of F-actin+/cortactin+ invadopodia structures in HCR cells (n=73 40x fields of view (FOV) across 3 biological replicates, points are average invadopodia/nuclei/FOV). G, Representative image of invadopodia rosettes (arrow with tail) seen in HCR-95 cells by staining with phalloidin (grey), Cortactin (green), and DAPI (magenta). H, Quantification of rosette number per nuclei, from live imaging of LifeAct transfected HCR cell lines (n=19-20 20x FOV across 2 biological replicates) I, Representative images of matrix degradation by each HCR cell line, seen as black puncta in regions where fluorescent gelatin plate coating was enzymatically degraded by invadopodia of overlying cells, J, Areas of mechanical matrix disruption below stress fiber adhesion points in HCR-95 (matrix – grey, degradation puncta - black, cortactin – green, DAPI – magenta pseudocolor). K, Quantification of matrix disruption area per nuclei (including both enzymatic degradation and mechanical disruption), displayed as log₂ fold change. Statistical significance was determined by one-way ANOVA with Kruskal-Wallis multiple comparisons test (B, C, H),Tukey’s multiple comparisons test (F), or Holm-Sidak multiple comparisons test (K). ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Error bars are represented as mean±s.e.m. Scale bars: 100 μm (A), 50 μm (I), 10 μm (D, E, G, J).