PIEZO2 cluster size and density is not affected by PKA modulation.A, representative TIRF images of N2A-P1KO cells transfected with PIEZO2mScarlet and incubated without (left, untreated) or with the PKA inhibitor KT5720 (middle) and the PKA activator 8-Br-cAMP (right). Scale bar is indicated in the images. B, average cluster densities of PIEZO2mScarlet in cells untreated (CTL, black or treated with KT5720 (gray) or 8-Br-cAMP (green). Data are presented as the mean ± SD with individual values. Number of cells is indicated in the graph. Comparison with one-way ANOVA, p = 0.5705. C, close-up view of the Gaussian fit of a PIEZO2mScarlet cluster (left) and average cluster size (in micrometer) per cell (right) and per treatment condition. Data are presented as the mean ± SD with individual values. Number of cells is indicated in the graph. Overall cluster number for CTL N = 3623, KT5720 N = 3792, and 8Br N = 5028. Scale bar is indicated in the image. Comparison with one-way ANOVA, p = 0.1066. See also Fig. S3. D, close-up view and representative examples (left) of the four different trajectories observed for PIEZO2mScarlet clusters: confined (cyan, top left), subdiffusion (orange, top right), normal diffusion (red, bottom left), and directed (green, bottom right). Average proportion of the four defined PIEZO2 cluster trajectories per cell (right). Data are presented as the mean ± SD. Number of cells are identical to the ones in B and C. Overall track numbers are identical to those in Fig. S3B. Scale bar represents 1 μm. Comparison with Kruskal–Wallis test, confined p = 0.4990, normal diffusion p = 0.1799, directed p = 0.5502, and subdiffusion p = 0.2792. E, representative fluorescent images (inverted) of N2A-P1KO cells untreated (control, left) or treated with the PKA inhibitor KT5720 (KT, middle) and the PKA activator 8-Br-cAMP (8Br, right). Scale bar is indicated in the images. F, quantification of the N2A-P1KO cell area (in μm2) incubated with the different PKA-modulated conditions. Data are presented as violin plot with the median value and the 25th and 75th quartile. Number of cells per group is CTL N = 1204, KT5720 N = 1637, and 8Br N = 939. Comparison with Kruskal–Wallis test p < 0.0001 and Dunn’s post-test, p = 0.00000065 CTL versus KT5720, p < 0.0001 KT versus 8Br, and p < 0.0001 CTL versus 8Br. 8-Br-cAMP, 8-bromo-cyclic-AMP; TIRF, total internal reflection microscopy.