Laminin-332 enhances the invasive potential of singles. (A) Live-cell tracking analysis of singles spheroids after treating with singles CM or leaders CM for 24 h (n = 15). Representative five tracks highlighted for each group. Scale bar, 50 μm. (B) Protein levels of laminin-332 in CM extracted from leaders with WT Lama3, and Lama3 CRISPR/Cas9 KO (clones C1 and D4). Total protein staining via Ponceau S was used as a loading control. Brightfield images were acquired after 24 h. Scale bar, 50 μm. (C) Live-cell tracking analysis of singles spheroids after treating with CM from WT Lama3 leaders and Lama3 KO leaders (clones C1 and D4) (n = 15). Representative five tracks highlighted for each group. Scale bar, 50 μm. (D) Live-cell tracking analysis of mCherry-transfected singles within a spheroid mixed 1:1 with mCherry-transfected singles and either leaders WT, clone C1, or clone D4 (n = 15). Representative five tracks highlighted for each group. Scale bar, 50 μm. For all experiments, three biological replicates were performed. For all panels: mean ± SEM is shown. Px/t stands for pixels/time, and px stands for pixels. Unless noted, n.s., no significance, *P ≤ 0.05, **P ≤ 0.01, ****P ≤ 0.0001. Source data are available for this figure: SourceData F4.