Fig. 1: Live imaging of epidermal cell behaviors in response to mechanical force.

a Schematic representation of the experimental setup and representative live imaging snapshots showing the formation of stress vesicles (yellow arrows) in the adult mouse epidermis following the application of negative pressure. Scale bar: 10 µm. b Quantification of the percentage of epidermal cells with stress vesicles as a function of the duration of applied negative pressure. Each point represents an individual mouse. Data from N = 3 mice (0 min, control), N = 3 (1 min), N = 5 (3 min), N = 6 (5 min), and N = 3 (10 min). Overall group differences were assessed using the Kruskal-Wallis test (P = 0.0012). Pairwise comparisons were performed as follows: unpaired t-test with Welch’s correction between 0 min and 1 min (P = 0.0016); one-way ANOVA followed by Šídák’s multiple comparisons test between successive timepoints: 1 min vs 3 min (P = 0.0217), 3 min vs 5 min (P < 0.0001), and 5 min vs 10 min (P = 0.0003). Data are presented as mean ± SEM. c Quantification of stress vesicle size with increasing duration of applied negative pressure. Each data point represents a single vesicle. A total of 753 vesicles were analyzed across all groups. A Spearman’s rank correlation test revealed a strong positive correlation between stress duration and vesicle size (r = 0.7324, 95% CI: 0.6963-0.7647, P < 0.0001). d Quantification of the percentage of cells exhibiting multiple stress vesicles as a function of the duration of applied negative pressure. Each data point represents an individual mouse. Overall group differences were evaluated using the Kruskal-Wallis test (P = 0.0019). Pairwise comparisons were conducted as follows: an unpaired t-test with Welch’s correction between 0 min and 1 min (P = 0.0286); and one-way ANOVA followed by Šídák’s multiple comparisons test between sequential timepoints: 1 min vs 3 min (P = 0.5490, not significant), 3 min vs 5 min (P = 0.0015), and 5 min vs 10 min (P = 0.1702, not significant). Data are shown as mean ± SEM. e Quantification of the percentage of deformed cell nuclei under varying durations of negative pressure. Each data point represents an individual mouse. Group differences were assessed using the Kruskal-Wallis test (P = 0.0001). Pairwise comparisons were performed using an unpaired t-test with Welch’s correction between 0 and 1 min (P = 0.0079), and one-way ANOVA with Šídák’s multiple comparisons test between successive timepoints: 1 vs 3 min (P = 0.0051), 3 vs 5 min (P < 0.0001), and 5 vs 10 min (P < 0.0001). Data are presented as mean ± SEM. f Quantification of nuclear volume in deformed nuclei under different durations of negative pressure. Quantification of nuclear volumes was performed across stress durations of 0 min (control), 1 min, 3 min, 5 min, and 10 min. Each point represents an individual nucleus, with a total of 444 nuclei analyzed. Spearman’s rank correlation analysis revealed a moderate inverse relationship between stress duration and nuclear volume (r = −0.3326; 95% CI: −0.4152 to −0.2446; P < 0.0001). g Representative timeframes from real-time imaging of stress vesicle growth in the basal and suprabasal cell layers of the mouse epidermis. Scale bar: 10 µm. h Quantification of the percentage of epidermal cells with stress vesicles as a function of the duration of applied negative pressure. Data from N = 4 mice; n = 16 images analyzed for each group. Statistical analysis was performed using a two-tailed unpaired t-test. i Representative frames at 3-s intervals of stress vesicle growth. Similar results were obtained from at least three independent experiments. Scale bar: 2 µm.