Fig. 4. β2 integrins respond differently to stiff and soft targets.
a, b BMDMs were challenged with 18 and 0.6 kPa IgG-coated DAAM particles and then subjected to phosphoproteomic analysis. a Schematic diagram of phosphoproteomic sample preparation—BMDMs and microparticles were co-incubated for 10 min before transferring to ice, and cells were then lifted and pelleted at −80 °C. b Volcano plot of the resulting phosphopeptides, with negative and positive log fold-change (LogFC) denoting enrichment with 18 and 0.6 kPa particles, respectively. LogFC and significance were calculated using 8 independent replicates for each condition. c Above: sequence of Itgb2758–773, with phosphorylated threonine highlighted in red. Below left, normalized abundance values of the Itgb2758–773 phosphopeptide. Below right, normalized abundance of all Itgb2 peptides without phospho-enrichment. n = 8 replicates (independent macrophage differentiations from three pooled mice). Error bars denote SD. P values determined by two-tailed t-test, with Bonferroni adjustment. ns not significant. d–f BMDMs were challenged with IgG-coated DAAM particles of different rigidities for various times, fixed and stained for F-actin and CD18, and then imaged by confocal microscopy. d Representative images of DAAM particle–macrophage conjugates after 5 min incubation, stained with anti-CD18 antibody (cyan) and phalloidin (F-actin, magenta). Rows 1 and 3: top–down maximum intensity projections, rows 2 and 4: side-view maximum intensity projections. Scale bars = 10 µm. CD18 accumulation (e) defined as the ratio of CD18 at the particle interface divided by total CD18 in the cell, and CD18 clearance index (f) comparing CD18 signal at the periphery and the center of the phagocytic cup (see “Methods” section) determined in phagocytic cups formed with 18 kPa (5 min) and 0.6 kPa (5 and 30 min) DAAM particles. In (e) and (f), error bars denote SD. P values determined by two-tailed Welch’s t-test. n = 58, 53, and 15 cells for 18 kPa, 0.6 kPa 5 min, and 0.6 kPa 30 min, respectively. Schematics in (a), (e), 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.