Fig. 2: 5-HT4R activation promotes actin polymerization in N1E-115 cells.
a Representative western blot (left) from the N1E-115 cells expressing 5-HT4R-eGFP after treatment with 5-HT for 10 min, subjected to the ultracentrifugation assay. The pellet fraction (P) contains the F-actin, while the supernatant fraction (S) is highly enriched with the G-actin. Right, the quantification of the relative F/G-actin ratio (N = 6 experiments for each group). *p < 0.05 (two-tailed unpaired t-test). b Confocal images for visualization of the F-actin fraction (F/(F + G)) in the N1E-115 cells expressing 5-HT4R-mCerulean treated with 5-HT, 5-HT + GR, and vehicle for 10 min. Scale bar: 10 µm. c Enlarged regions of interest (left), selected as white boxes in (b) where analysis of the actin polymerization was performed. Array of lines perpendicular to the plasma membrane was automatically generated, along those the analysis of the F-actin staining from outside to inside the cell was determined, Scale bar: 10 µm. Right, quantification of the F-actin staining intensity. d Changes in the F/G-actin ratio calculated for the entire cell (N = 4 experiments for each group, at least 8 cells analyzed per condition). **p < 0.01, ***p < 0.001 (two-way ANOVA, post hoc Tukey test). e Representative confocal images (left) to visualize the F-actin fraction (F/(F + G)) in N1E-115 cells co-transfected with 5-HT4R-mCerulean and either scr-shRNA or anti-Gαs shRNA, or anti-Gα13 shRNA and treated with 5-HT or vehicle for 10 min. Scale bar, 10 µm. Right, quantification of the relative changes in the F/G-actin ratio (N = 6 experiments for each group with at least 7 cells analyzed per condition). **P < 0.01, ***P < 0.001 (two-way ANOVA, post hoc Sidak test). See also Supplementary Fig. 3. All data are presented as mean ± SEM.
