3D Cell Culture and Cytoskeleton Imaging

For 3D cultures, gels were prepared as previously described^{18 (link)}. Briefly, 1.5 × 10⁴ FRCs were mixed with 100 µL of the Matrigel mix (1.8 mg/ml reduced growth factor Matrigel (BD Biosciences) and 3.2 mg/ml type I collagen (BD biosciences)) and plated in glass-bottom 24-well plates (MatTek Corporation). For treatment conditions, a final concentration of 10 µg/mL anti-PDPN (Biolegend, clone 8.1.1), 7 µg/mL CLEC-2-Fc (made in-house^{18 (link)}), or appropriate controls were added to the cell suspensions 15 m before the addition of the Matrigel mix. For short-term treatments, the cells were allowed to populate the gels for 24 h and then 250 nm cytochalasin D (Sigma), 10 µm Y27632 (Sigma), or 7 µg/mL CLEC-2-Fc were added directly to the media. To image the cells, the media was removed and the gels were washed twice in PBS. Next, cells were fixed in 4% paraformaldehyde (PFA; Affymetrix), washed, and permeabilized in 0.25% Triton-X. Cells were then stained with DAPI and rhodamine phalloidin (Invitrogen). The gels were washed with PBS and imaged with a Leica SP5X laser-scanning confocal microscope. z-stack projections were generated in ImageJ (imagej.nih.gov), and the area and perimeter of the cells were measured. The morphology index was calculated with the following equation: perimeter² /4*π*area.