Fig. 3: Stiffness-dependent Piezo1 expression/activity modulates macrophage activation.

a Representative Western blots (left) and quantification (right) of iNOS, ARG1, and GAPDH of BMDMs seeded on 1, 20, 40, and 280 kPa polyacrylamide gels and incubated with media (Unstim.), 0.3 ng/mL IFNγ/LPS, or 0.1 ng/mL IL4/IL13. Data normalized to the 1 kPa IFNγ/LPS condition. Samples were derived from the same experiment and the resulting blots were processed in parallel. b, c Representative Western blots (top) and quantification (bottom) of p-NFκB/NFκB (b) and p-STAT6/STAT6 (c) in wild type BMDMs seeded on 1 and 280 kPa polyacrylamide gels and stimulated with IFNγ/LPS or IL4/IL13, respectively. Phosphorylated and total forms of transcription factors were obtained from loading equal amounts of protein in separate gels and the resulting blots were processed in parallel. d Representative immunofluorescent images (left) and quantification of fluorescence (right) of Piezo1^P1-tdT BMDMs seeded on polyacrylamide gels, tdTomato is displayed in red. Quantification of e number of Ca²⁺ events (normalized for cell number and time) and f fraction of cells showing Ca²⁺ elevations in unstimulated BMDMs seeded on 1, 20, 40, and 280 kPa polyacrylamide surfaces following acute addition of Ringer solution (Unstim.) or Ringer solution with 100 ng/mL IFNγ/LPS, captured by confocal microscopy. Each data point is calculated from a 10-min time-lapse video (N = 6 for each stiffness). g Representative G/R ratio images (left) and quantification of peak G/R intensities of BMDMs seeded on polyacrylamide gels of indicated stiffness and exposed to DMSO, 300 nM Yoda1, and 5 µM Yoda1. N = 107, 248, 145 cells on 1 kPa, n = 118, 239, 169 cells on 20 kPa, n = 113, 250, 183 cells on 40 kPa, and n = 106, 274, 184 cells on 280 Pa and exposed to Rest, 300 nM Yoda1, or 5 µM Yoda1, respectively. Data representative of three independent experiments. h Relative Nos2 gene expression of BMDMs seeded on surfaces of indicated stiffness, exposed to DMSO, 300 nM Yoda1, or 5 µM Yoda1, and stimulated with 0.3 ng/mL IFNγ/LPS. Data normalized to 1 kPa DMSO control. i Relative Arg1 gene expression of BMDMs seeded on surfaces of indicated stiffness, exposed to DMSO, 300 nM Yoda1, or 5 µM Yoda1, and stimulated with 0.1 ng/mL IL4/IL13. Data normalized to 1 kPa DMSO control. j Relative Nos2 gene expression Piezo1^fl/+ and Piezo1^ΔLysM BMDMs seeded on surfaces of indicated stiffness and stimulated with 0.3 ng/mL IFNγ/LPS. Data normalized to 1 kPa Piezo1 control. k Relative Arg1 gene expression of Piezo1^fl/+ and Piezo1^ΔLysM BMDMs seeded on surfaces of indicated stiffness, and stimulated with 0.1 ng/mL IL4/IL13. Data normalized to 1 kPa Piezo1^fl/+ controls. Error bars denote Mean ± SD for three separate experiments, * p < 0.05 as determined by two-tailed paired t test (a, e, h–k), two-tailed Student’s test (b–d), or Mann–Whitney U test (g). Source data including exact p-values are provided as a Source Data file.