Fig. 7. Talin1 and Vinculin mediated mechanosensing is required for enhanced uptake of stiff cargo.

a, b Cas9⁺ HoxB8-ER conditionally immortalized progenitors were transduced with either a non-targeting sgRNA or sgRNA targeting Talin1 or Vinculin, and then differentiated into macrophages. These cells were then challenged with 10 pmol/M IgG-coated 18 kPa or 0.6 kPa DAAM particles. Graphs show phagocytic efficiency normalized against the sgNT, 0.6 kPa sample. n = 3 biological replicates for each genotype. P values determined by two-tailed paired ratio t-test. ns not significant. c F-actin coverage after 30 min incubation, determined for Talin1 and Vinculin CRISPR KOs (sgTalin1, sgVinculin) and non-targeting controls (sgNT). Values are sorted into five bins, with colors denoting the fraction of events falling within each bin. Representative bars from one biological replicate are shown, from 18 (sgVinculin) or 20 (sgNT and sgTalin1) events/condition. d Fraction of events that are 20–95% complete after 30 min co-incubation of 100 pmol/M IgG-coated DAAM particles with HoxB8-derived macrophages. n = 3 replicate differentiations (15–20 events/condition). P values determined by two-tailed paired t-test. e Representative images of BMDM-particle interactions showing Vinculin (yellow) and phalloidin (F-actin, magenta) staining, along with DAAM particles in green. Each panel contains top views (right) and side views (left). Scale bars = 5 µm. f Vinculin accumulation (see “Methods” section) at phagocytic cups, graphed as a function of particle stiffness and Itgb2 genotype. From left to right, n = 47, 78, 41, 47 cell–particle conjugates measured over three independent experiments. P values determined by Welch’s two-tailed t-test. ns not significant. All error bars denote SD. Schematics in (a) and (f) created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en.