PDCD4 axonal levels change during neuron development and modulation of this protein can control axonal growth. (A) Immunocytochemistry assays show that PDCD4 levels increase during cortical primary neurons differentiation in vitro (scale bar, 20 µm). Cell bodies were indicated by filled arrows and axons by unfilled arrows. The signal quantification shows that cell bodies and axons have a significant increase in PDCD4 expression for day 5, and a trend to increase for day 12, always compared to day 2 ([***] P ≤ 0.001, ANOVA test with post-Tukey, error bars: SEM, n = 3 independent primary cortical neuron cultures for day 2 and day 5, with three technical replicates for each independent experiment; n = 2 for day 12, with two technical replicates for each independent experiment). (B) Cotransfected cortical primary neurons with a GFP plasmid and a PDCD4 plasmid, or a GFP plasmid and a pcDNA plasmid (scale bar, 20 µm). Overexpression of PDCD4 in transfected neurons at day 5 induce a decrease in axonal length (25%) compared to control condition ([**] P ≤ 0.01, paired test, n = 5 independent primary cortical neuron cultures, error bars: SEM). (C) Same as above but for PDCD4 knockdown using an siRNA for PDCD4 or an siRNA control (scale bar, 20 µm). Down-regulation of PDCD4 induces an increase in axonal length (18%) compared to the control condition ([**] P ≤ 0.01, paired t-test, n = 6 independent primary cortical neuron cultures, error bars: SEM). (D) Immunocytochemistry assays with acetylated tubulin in peripheral DRG neurons cultured in compartmentalized chambers and transfected with a permeable siRNA for PDCD4, or with a siRNA control (scale bar, 500 µm). Quantification of axonal growth shows similar effect as above: down-regulation of PDCD4 determines an increase (24%) of axonal growth increase (* P ≤ 0.05, Student's t-test, n = 4, error bars: SEM). In all cases, the “n” corresponds to independent biological replicates.
