Effects of the down-regulation of AKAP79 on TRPV4 activity. (A) Representative two-colored super-resolution images generated using GSD in control tsA-201 cells (left) and AKAP79-siRNA–treated cells (right) stained with anti-AKAP79 and anti-TRPV4 antibodies followed by Alexa Fluor 568– and Alexa Fluor 647–labeled secondary antibodies, respectively. Composite and mask images to the right show the boxed regions at higher zoom. (B) Two histograms representing the frequency of distances between TRPV4 and AKAP70 proteins in control conditions (left, gray) and in AKAP79-siRNA–treated cells (right, red). Solid lines represent best-fit Gaussian distribution. TRPV4-AKAP distance means are 155.3 ± 5.25 nm in control cells and 352.1 ± 7.36 nm in AKAP-siRNA cells. (C) Bar plot showing TRPV4, AKAP79, and overlap mean ± SEM areas (nm2) in control conditions and under presence of AKAP79-siRNA. (D) Real-time PCR analysis of mean ± SEM diffrence of AKAP79 mRNA in control, mock, and AKAP79-siRNA–treated tsA-201 cells. (E) Representative TIRF images of TRPV4-EGFP in control (top) and AKAP79-siRNA–treated (bottom) tsA-201 cells. Bar plot of mean ± SEM TRPV4-EGFP fluorescence in control and AKAP79-siRNA–treated tsA-201 cells (right). A.U., arbitrary units. (F) Representative ITRPV4 records from a control and AKAP79-siRNA–treated cell. (G) Bar plot of the mean ± SEM amplitude of ITRPV4 at 80 mV in control and AKAP79-siRNA–treated cells. (H and I) Mathematical simulation of the effects of down-regulating AKAP150 (H) or up-regulating TRPV4 (I) on TRPV4 channel activity in male and female myocytes. **, P < 0.01; ***, P < 0.001.
