Fig. 3. Knockdown of the P2X7 receptor promotes axon development. (A) Western blotting of HEK 293T cells, and of HEK 293T cells transfected with shRNA-Luc plus pcDNA-P2X7 or with shRNA-P2X7 plus pcDNA-P2X7. The levels of -tubulin were used as a loading control and the P2X7:-tubulin ratio was used to estimate the efficiency of the selected shRNA-P2X7. Histogram values were normalized to the value of shRNA-Luc plus pcDNA P2X7-transfected HEK 293T cells (n=3, ***P<0.001). (B) Representative GFP-fluorescence images of hippocampal neurons transfected at 1 DIV with pEGFP, shRNA-Luc or shRNA-P2X7. Neurons were fixed and their axon length and ramifications were analyzed at 3 DIV. Scale bar: 25 µm. (C) Graphs represent the mean ± s.e.m. of the axon length and their ramifications in each neuron from three different experiments (n=60; ***P<0.001, two-way ANOVA). (D) Hippocampal neurons nucleofected with the GFP, shRNA-P2X7 or P2X7-GFP expression plasmids. Neurons were nucleofected before plating and kept in culture for 3 days. Neurons were fixed and stained for tyrosinated -tubulin (red) and GFP-expressing neurons were identified as nucleofected neurons (green). Scale bars: 50 µm.