IRSp53 depletion mimics aspects of the Par1b overexpression phenotype. (A) Dox-inducible IRSp53 depletion is shown. (B) Brightfield time lapse of IRSp53-KD cell islands on collagen I (Video 10). (C) Polarized cultures 24 h after Ca²⁺ switch. Phalloidin labels stress fibers at the basal domain (bottom) and microvilli-rich lateral lumina and apical domains in the maximum (max) projections (top; see Video 7). (D and F) Still images from time-lapse videos taken 30 min after plating on collagen. (D) Brightfield and mRFP fluorescence (Video 8). (F) Paxillin-mCherry (Video 9, top) is shown. Arrowheads point to FAs. (E) Spreading of two IRSp53-KD clones on collagen I in DME and SMEM; all differences between ±dox are significant with P < 0.001. Error bars indicate SEM. (G) Confocal section through collagen cysts (see Video 4 for full stacks) annotated as in Fig. 1 G. IRSp53KD cysts are smaller than control cysts (14 vs. 25 cells/cyst; n = 31). Bars: (B–D and F) 10 µm; (G) 25 µm.