Rac1-GTP localizes to the periphery in mesenchymal clusters and polarizes internally in epithelial-like clusters during SDF1 chemotaxis. (A) Immunostaining of active Rac1 (Rac1-GTP) in mesenchymal and epithelial-like NC clusters. Scale bar: 100 µm. (B) Corresponding fluorescence heatmaps showing Rac1-GTP distribution. (C and D) Zoom 1 highlights the cluster periphery; Zoom 2 shows the internal region. Quantification of Rac1-GTP fluorescence intensity in mesenchymal (C) and epithelial-like (D) clusters in the absence of chemotactic cue, comparing peripheral and internal regions (n = 9 per region per condition). (E) Immunostaining of Rac1-GTP in mesenchymal and epithelial-like clusters during chemotaxis to SDF1. Scale bar: 100 µm. (F) Heatmaps of Rac1-GTP signal during chemotaxis. Zoom 1 and 2 show peripheral and internal cluster regions, respectively. (G and H) Quantification of Rac1-GTP fluorescence intensity in mesenchymal (G) (n = 8) and epithelial-like (H) (n = 9) clusters during chemotaxis. Each dot represents one cluster. Data are from at least three independent experiments. Error bars show mean ± SEM. Error bars show mean ± SEM. Statistical analysis was performed using unpaired two-tailed Student’s t test. ****P ≤ 0.0001; *P ≤ 0.05.