Figure 5.

Prg2 Overexpression in ES-Cell-Derived Neurons Induces Axon Filopodia Involving PI3K/PI(3,4,5)P₃

(A) Induction of Prg2-Flag expression after doxycycline (DOX) application. Lysates of ES-cell-derived motor neurons (ESCMNs) treated with 2 μg/mL doxycycline for 24 h were analyzed using the indicated antibodies.

(B) ESCMN culture scheme.

(C) Images show representative examples of neurons stained with Phalloidin (F-actin) after Prg2 induction with or without the PI3K inhibitor LY294002 (15 μM, 2 h). Scale bar: 10 μm. Doxycycline-induced Prg2 expression results in an increase in the number of axon filopodia. The PRG2-induced effect is reversed by PI3K inhibition using LY294002.

(D) Quantification of filopodia. Bars show the mean number of filopodia per neuron, and the average length of filopodia/neuron ± SEM of 2–3 independent experiments performed in triplicates, n ≥ 100. **p < 0.01, ^∗∗∗p < 0.001 (one-way ANOVA with Bonferroni post hoc test).

(E) Prg2-induced ESCMNs cultured for 24 h in the presence or absence of LY294002 (15 μM). PRG2 increases the number of axons and axon branches in dependence of PI3K/PI(3,4,5)P₃. Cells were stained with anti-tubulin antibody (green) and Phalloidin (red). Scale bar: 30 μm.

(F) Quantification of axons per cell and axonal branching. Bars show the mean number of branches ± SEM of two to three independent experiments performed in triplicates, n ≥ 120. ∗∗p < 0.01, ^∗∗∗p < 0.001 (one-way ANOVA with Bonferroni post hoc test).

See also Figure S4.