Fig. 1: 5-HT4R activation increases cofilin phosphorylation, RhoA activity, and neurite retraction in the G13-dependent manner.
a N1E-115 cells transfected with pcDNA or 5-HT4R-eGFP were treated for 5 min with 5-HT, BIMU8 alone or in combination with 5-HT4R antagonist GR, or vehicle (veh), followed by the detection with antibodies against phosphorylated cofilin (p-Cof), total cofilin (Cof), and GAPDH (bottom) and quantification of the relative cofilin phosphorylation in N1E-115 cells (right). N = at least three experiments for each group. See also Supplementary Fig. 1. b Representative western blots (left) to visualize cofilin phosphorylation in N1E-115 cells co-transfected with 5-HT4R-eGFP and shRNA against Gαs (shGαs) or Gα13 (shGα13), or scrambled shRNA (scr) and treated with 5-HT or vehicle (veh). Quantification of cofilin phosphorylation (right). N = 5 experiments for each group. See also Supplementary Fig. 1. c Representative western blots (left) to visualize cofilin phosphorylation in N1E-115 cells transfected with 5-HT4R-eGFP either alone or in combination with WT or dominant-negative RhoA mutant and treated with 5-HT. Quantification of the relative cofilin phosphorylation (right) in N1E-115 cells (N = 4 experiments for each group). See also Supplementary Fig. 1. d Time-lapse confocal ratiometric images of the N1E-115 cells co-transfected with 5-HT4R-mCherry together with FRET-based biosensor Raichu-RhoA. After 5 min imaging under control conditions, either vehicle or 5-HT was added to the bath solution and cells were imaged for the next 15 min. Increase in the YPet/mTurquoise ratio is equivalent to an increase in the RhoA activity. Scale bar: 20 µm. Lowest row shows the enlargement corresponding to the white box. Scale bar: 10 µm. e Quantification of the RhoA activity expressed as a YPet/mTurquoise ratio within the whole cell over time (N = 6 cells for each group, at least 3 independent experiments were conducted). See also Supplementary Fig. 2. f Contours of cells treated with vehicle (veh) or 5-HT at different time points. See also Supplementary Fig. 2. All data are presented as mean ± SEM. *p < 0.05; **p < 0.01, ***p < 0.001 (two-way ANOVA with Sidak test; a–c). *p < 0.05, **p < 0.01 (Mann–Whitney test; e).
