Fig. 2. Chemical inhibition of neuronal activity selectively regulates the assembly of cytoplasmic DDX6 granules in mature hippocampal neurons.

a Representative examples of DDX6 immunostainings and phase contrast pictures of 22 DIV hippocampal neurons in culture under vehicle (DMSO) treated or silenced (100 µM CNQX, 50 µM AP5, 1 µM TTX) conditions. Boxed regions in images are displayed as magnified insets. Scale bar 10 µm. b, e Bar plots displaying quantification of cell population by fraction of cells containing either large or small DDX6 granules as exemplified in a under untreated, vehicle treated, or silenced conditions (b, e), followed by recovery or NMDA treatment (e). Experimental outline is presented in e. Data represents mean ± standard deviation of three independent neuronal cultures. Distinct dot symbols indicate biological replicates. At least 100 cells/condition/experiment were quantified. c, d Dot plots displaying average DDX6 granule size (c) and DDX6 granule number (d) of individual cell bodies under vehicle treated (DMSO) or silenced conditions. Small gray symbols represent single cells while larger white symbols indicate the average of each replicate. Horizontal line and error bars represent mean of replicates and standard deviation (n = 4 biologically independent experiments). Asterisks represent p-values obtained by two-sided Student’s t-test (b–d) or Tukey’s test post-hoc to two-way ANOVA analysis (e) (**p < 0.01). Hashtags represent p-values obtained by Tukey’s test compared to untreated conditions (e) (###p < 0.001). p = 0.0012 (b), p = 0.00053 (c), p = 0.00010 (d), F_2,18 = 1.59e-05, treatment 1, F_2,18 = 2.38e-11, treatment 2 (e).