Fig. 4: PIEOZ1 activity directs stem cell-like states and tumorigenicity after microconstriction.

A Representative fluorescence images of PIEZO1 expression for CTRL and SQZD cells. B Quantification of PIEZO1 immunofluorescence intensity expressed as the mean ± SEM from five independent experiments. Statistical significance assessed using a two-sided unpaired t-test: p  = 0.0003 (***). p  =  0.0003(***), 95% CI: [8.521, 18.99]. C Representative fluorescence images showing melanoma cells as they squeeze through the constrictive channels with quantification of fold change intracellular calcium concentration using Calbryte 520 AM. The results are expressed as the mean ± SEM from 2 independent experiments. Statistical significance was assessed using one-way ANOVA (two-sided): 30 µm vs. 10 µm (CC) p  =  0.0185 (*) 95% CI: [−0.1084 to −0.01587]; 30 µm vs. 5 µm (CC) p = 0.0071 (**) 95% CI: [−0.1271 to −0.03463]. D Representative fluorescence images of cancer cells showing stemness biomarkers under CTRL condition and after treatment with 20 µM Yoda1 and 30 µM Ruthenium Red (RR). E Bar graph showing the fold change of fluorescence signal of stemness-related biomarkers for cells under different treatments. The result is expressed as the mean ± SEM from three independent experiments. Statistical significance was assessed using a two-sided unpaired t-test. Asterisks indicate p-values as follows: CD44 p  =  0.0044 (**) 95% CI: [−0.9418, −0.3318], PRDM14 p  =  0.0212 (*) 95% CI: [−1.228, −0.1722], CD271 p  =  0.0175 (*) 95% CI: [−2.537, −0.4282], ABCB5 p  =  0.048 (*) 95% CI: [−5.333, −0.02600]. F-H Images of CTRL and treated melanoma cells, forming tumorsphere in serum-free media after 7 days of culture, demonstration of increased mean tumorsphere area over time and quantification of tumorsphere area expressed as the mean ± SEM from four independent experiments, except for YODA (n = 3). Statistical significance was assessed using two-way ANOVA (two-sided). Asterisks indicate p-values as follows: CTRL vs. YODA p  =  0.0346 (*) 95% CI: [−2712, −114.6]; CTRL vs. RR p  =  0.0333 (*) 95% CI: [117.0, 2522]; YODA vs. RR p  =  0.0008 (***) 95% CI: [1434, 4032].