Fig. 2.

CPC reduces the PM/Cytosolic ratio of PIP₂-binding protein MARCKS in RBL-2H3 cells and reduces the mean density of the PIP₂-binding protein PAmKate-PH in PM of NIH-3T3 cells. (A) Representative live-cell confocal microscopy images of RBL-2H3 cells expressing mRFP-MARCKS-ED, ± CPC exposure: Control (0 μM CPC), 5 μM CPC, or 10 μM CPC for 30 min. After washing off the CPC with BT, confocal images were taken. (B) The ratio of the mean fluorescence per pixel of MARCKS at the PM and at the cytosol. Values represent mean ± SEM of three independent experiments that were derived from analysis of n = 143 cells for Control (0 μM CPC), n = 90 cells for 5 μM CPC, and n = 65 cells for 10 μM CPC. (C) Quantification of PH levels at the PM using super-resolution microscopy. NIH-3T3 cells expressing PAmKate-PH, ± CPC exposure for one hour, then fixed by 4% PFA. FPALM imaging of the PM of the transfected cells was carried out with TIRF excitation. The mean density of PH molecules was plotted as a function of CPC treatment. Values represent mean ± SEM of three independent experiments from analysis of n = 32 cells for BT Control (0 μM CPC), n = 30 cells for 5 μM CPC, and n = 31 cells for 10 μM CPC. Statistically significant results, as compared to Control (0 μM CPC), are represented by *p < 0.05 and **p < 0.01, as determined by one-way ANOVA followed by Dunnett's post-test.