Figure 9. Yoda1-induced calpain2 activation promotes profibrotic responses in HK2 cells.

(A and B) Fluo-4-AM was added to HK2 cells treated with Yoda1 for 1 hour, 3 hours, 6 hours, and 24 hours, and the fluorescence signals of Ca²⁺ were detected by flow cytometry analysis and fluorescence microscope. Scale bar: 200 μm. “50% Show” means that flow cytometry data are presented based on 10,000 cells of 20,000 cells analyzed. (C and D) Representative immunoblots and corresponding densitometry analysis of calpain2, fibronectin, α-SMA, and E-cadherin protein abundance in HK2 cells pretreated with 2 mM EGTA followed by Yoda1 treatment for 24 hours. Data are shown as mean ± SEM (n = 4 in each group). *P < 0.05 when compared with CTL and ^#P < 0.05 when compared with Yoda1 by 1-way ANOVA with Student-Newman-Keuls test. (E and F) Representative immunoblots and corresponding densitometry analysis of calpain2 protein abundance in HK2 cells transfected with Piezo1 siRNA followed by Yoda1 treatment for 24 hours. Data are shown as mean ± SEM (n = 4 in each group). *P < 0.05 when compared with scramble siRNA CTL and ^#P < 0.05 when compared with scramble siRNA Yoda1 by 1-way ANOVA with Student-Newman-Keuls test. (G and H) Representative immunoblots and corresponding densitometry analysis of calpain2, fibronectin, α-SMA, and E-cadherin protein abundance in WT HK2 cells and CAPN2-KD HK2 cells treated with Yoda1 for 24 hours. Data are shown as mean ± SEM (n = 4 in each group). *P < 0.05 when compared with CTL by 1-way ANOVA with Student-Newman-Keuls test. CAPN2-KD, calpain2 knockdown.