Fig. 6: RGD-binding integrin-specific cell spreading and force transmission.

a Binding affinities of cRGDfK peptide to intact RGD-binding integrins on K562 cells in L15 medium with or without 1 mM Mn²⁺ and to the EO states of integrin α5β1 and αVβ1 stabilized by 12G10 Fab. Titration curves are shown in Supplementary Fig. 9. b Integrin expression on BJ-5ta cells quantified by dose dependent staining with human IgG1s and fluorescent goat anti-human IgG. Background signal with non-binding IgG1 is subtracted. Binding was fitted to dose response curve to obtain antibody EC50 and maximum specific mean fluorescence intensity (MFI). c K_d values of RGD-mimetic Fabs against cell surface RGD-binding integrins. Measurements were either with or without 1 mM Mn²⁺ (Supplementary Fig. 10). Not bound: no significant binding up to 10 µM. d Percentage of Fab-unbound RGD-binding integrins in presence of specific inhibiting Fabs or combinations of them. Percentage of Fab-unbound integrin subtype is calculated based on \({{{{{{\rm{P}}}}}}}^{{{{{{\rm{Fab}}}}}}-{{{{{\rm{unbound}}}}}}}=\mathop{\sum}\nolimits_{{{{{{\rm{i}}}}}}}\frac{1}{{1+{{{{{\rm{C}}}}}}}_{{{{{{\rm{Fab}}}}}},{{{{{\rm{i}}}}}}}/{{{{{{\rm{K}}}}}}}_{{{{{{\rm{d}}}}}},{{{{{\rm{i}}}}}}}}\), where C_Fab,i is the concentration used for i^th Fab, and K_d,i is the K_d value of ith Fab to the specified integrin subtype. IPI Fabs shown in panel c and mAb16 Fab to α5β1 were at 5 µM; Biogen-αVβ1.5 Fab was at 10 µM. Affinities of Fabs measured without Mn²⁺ (panel c) were used to calculate the percentage of Fab-unbound integrins. e–i BJ-5ta spreading in presence of integrin blocking Fabs as described in panel d legend. Cells were pre-incubated with Fabs for 5 min and seeded for 1 h on each surface. e Integrin αVβ1 dependent cell spreading on RGD-54pN (mean ± SE for three independent experiments, n = 26–101 cells for each experiment). f Close-contact enclosed area of cells on RGD-54pN analyzed from RICM images (mean ± SE; n = 43, 90, 53, 58, 81, 121, 83, 76, 102, 43 cells). g Aspect ratio of the area (mean ± SE; n = 33, 34, 37, 32, 61, 48, 26 cells; >1000 μm²). h Immunostaining of paxillin (green, AF488) and actin stress fibers (red, SiR-actin). Cells were fixed after 1 h spreading on RGD-54pN. Scale bars: 10 µm (white), 2 µm (yellow). Boxed regions in the top row were magnified in the bottom row. i Cells were seeded on RGD-54pN with BHQ2-Cy3 and the ruptured TGT was quantified (mean ± SE; n = 46, 41, 27, 35, 36, 65 cells). Two-sided t-test for p-values. Images are representative of multiple experiments and cell data points are combined from three independent experiments. Source data are provided as a Source Data file.