Depletion of CD9 and CD81 promotes cell migration on various ECM substrates. MCF7 cells with simultaneous knockdown of CD9 and CD81 (pLVHTM-CD9 shRNA + CD81 siRNA, CD9/CD81 dKD MCF7; control cells transfected with pLVTHM-EGFP and negative control siRNA, Ctrl MCF7) were seeded as single cell suspension on µ-Dishes coated with different ECM substrates. Cell migration was observed using digital holographic microscopy with a 20× objective (A) or using the confocal LSM780 microscope (Zeiss) equipped with a 20× objective (C and D) over 50 h. Tracking of individual cells was performed as described in the Methods section. (A) Representative images of migration tracks of Ctrl MCF7 cells and CD9/CD81 double KD MCF7 cells grown on VN. (B–D) Accumulated Distance and Euclidean Distance of Ctrl MCF7 cells and CD9/CD81 double KD MCF7 cells grown on VN (B; ctrl MCF7: n = 51, CD9/CD81 KD MCF7: n = 50), LN (C; ctrl MCF7: n = 61, CD9/CD81 KD MCF7: n = 62), or FN (D; ctrl MCF7: n = 55, CD9/CD81 KD MCF7: n = 72). Data is derived from three independent experiments and is represented as mean ± SD. Statistical significance was calculated using Mann–Whitney U test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.