Figure 5.

Stiffness-dependent Piezo1 expression/activity modulates microglia migration and pro-inflammatory cytokines production

(A) Representative images of migrated BV2-WT or Piezo1-KO cells pre-cultured for 48h in PA gel of varying stiffness (Soft: 0.6 KPa, Stiff: 35 KPa) stained by crystal violet. Scale bar = 100um.

(B) Quantification of migrated cell numbers of BV2-WT or Piezo1-KO cells pre-cultured for 48h in PA gel of different stiffness (Soft: 0.6 KPa, Stiff: 35 KPa). Bars represent mean ± SD N = 3 independent experiments. two-way ANOVA, n.s., no significance, ∗p < 0.05, ∗∗p < 0.01.

(C-E) Relative IL-1β, IL-6, and TNF-α mRNA expression of BV2-WT and BV2-Piezo1 KO seeded on surfaces of indicated stiffness (Soft: 0.6 KPa, Stiff: 35 KPa). Bars represent mean ± SEM N = 5 independent experiments. two-way ANOVA, n.s., no significance; ∗p < 0.05, ∗∗∗∗p < 0.0001.

(F) Immunofluorescence confocal images of Piezo1 (green) in BV2 cell lines seeding on different stiffness of PA gel (Soft: 0.6 KPa, Stiff: 35 KPa). Scale bar = 20 μm

(G) Quantification of Piezo1 mean fluorescent intensity (MFI) of BV2 cells seeding on different stiffness of PA gel. Bars represent mean ± SD Repeated 3 times, around 30 cells were analyzed for each experiment. Two-tailed unpaired t test. ∗∗∗∗p < 0.0001.

(H) Relative Piezo1 mRNA levels of BV2 cell lines seeding on different stiffness of PA gel. Bars represent mean ± SEM N = 4 independent experiments. Two-tailed unpaired t test. ∗p < 0.05.

(I) Representative live-cell imaging of BV2 cells loaded with Fura2 calcium indicator treated with Yoda1 (10 μM) at time 0 (left panel) and at the time of maximum fluorescence (right panel) seeding on soft and stiff substrate respectively. Scale bar = 20 μm

(J) Representative calcium imaging traces of Fura-2 (F340/F380 ratio) in BV2 cells seeding on soft (black curve) and stiff substrates (red curve) treated with Yoda1 (10 μM).

(K) Statistical analysis of ΔF/F0 (%) of BV2 cells seeding on soft (black) and stiff substrates (red) induced by Yoda1 (10 μM). Bars represent mean ± SD N = 3 independent experiments, 20-30 cells were analyzed for each experiment. Two-tailed Mann-Whitney U test. ∗∗∗∗p < 0.0001.