Supplementary Figure 3: Validation of reticular adhesion independence from F-actin and talin. | Nature Cell Biology

Supplementary Figure 3: Validation of reticular adhesion independence from F-actin and talin.

From: Reticular adhesions are a distinct class of cell-matrix adhesions that mediate attachment during mitosis

Supplementary Figure 3

(a, upper) Confocal images of U2OS cells attached to VN-coated surfaces and labelled for integrin β5, vinculin and F-actin, following 30 min in suspension and 3 h replating in the presence of DMSO, 20 µM CytoD, or 1 µM latrunculin A (LatA). (a, lower) Representative confocal images of U2OS cells attached to vitronectin (VN)-coated surfaces and labeled for integrin β5, vinculin and F-actin, following 3 h replating, with final 1 h in presence of DMSO or 20 µM CytoD. (b) Images comparing attachment of CS1WT or CS1β5 cells to vitronectin in the presence of RAD or RGD peptides and/or CytoD. Zoomed panels demonstrate image-based identification of attached cells using automated cell counting in NIS Elements. Representative of n = 6 biologically independent experiments. (c) Representative immunoblot of talin-2 (68e7 antibody) and tubulin following siRNA treatment with control (CTRL) oligonucleotides or one of three alternative talin-2-targeting oligonucleotides. Unprocessed blots in Supplementary Fig 9. (d) Confocal images of U2OS cells expressing RFP-talin head or RFP-talin rod domains plated on glass coverslips for 48 hours and immunolabeled for integrin β5 and vinculin. Images in A, C-D representative of results from at least n = 3 biologically independent experiments. Scale bars: A, Bvi-viii = 10 µm; Bi-ii and Bix-x = 1000 µm; Biii-v = 100 µm; D = 5 µm

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