Figure 5

Involvement of KCa3.1 in inhibitory effect of Yoda1 on ruffle formation. (A) Live cell imaging of EGF-induced membrane ruffles in A431 cells. The cells were pretreated with a KCa3.1 inhibitor TRAM-34 for 5 min and then stimulated with EGF in the presence of Yoda1 (1.5 µM). Live cell imaging was started immediately after adding EGF and Yoda1. DIC images at the indicated time points (0, 2.5 and 5 min) are shown. The right image shows an enlarged view of the area outlined by the white square in the image at 5 min. (B) F-actin staining with phalloidin. Left: Representative images of A431 cells pretreated with TRAM-34 for 5 min and then stimulated with EGF in the absence or presence of Yoda1 (1.5 µM) for 5 min. Right: Quantification of cells with peripheral ruffles. (C) F-actin staining with phalloidin. Left: Representative images of A431 cells pretreated with KCa3.1 activator SKA-31 for 5 min and then stimulated with EGF for 5 min. Right: Quantification of cells with peripheral ruffles. (D) Flow cytometry analysis of TMR-dex70 uptake. The cells were pretreated with KCa3.1 activator SKA-31 for 5 min and then stimulated with EGF for the uptake of TMR-dex70 for 10 min. (E) Observation of TMR-dex70 uptake. The cells were treated as in (D). (F) Working hypothesis of macropinocytosis inhibition using Yoda1. Yoda1-induced, Piezo1-dependent extracellular Ca²⁺ influx causes non-proper activation of KCa3.1, which inhibits Rac1 activation followed by ruffle formation. PowerPoint (version 16.57, Microsoft) was used to generate the picture. Data in (B) and (C) represent the mean ± s.e.m. (number of total counted cells pooled from three independent experiments: from left to right, (B) 540, 559, 562, and 552; (C) 538 and 561). Data in (D) represent the mean ± s.e.m. (n = 3 independent biological replicates). *p < 0.05; ***p < 0.001; n.s. not significant [one-way ANOVA followed by Tukey–Kramer’s post hoc test (B,D) or Student’s t test (C)]. Yellow arrowheads in (B) and (C) indicate F-actin positive peripheral membrane ruffling area. Scale bars, (A,E) 20 µm; (B,C) 50 µm.