Figure 9.

Characterization of DLC1, ARHGAP5, and double-KD phenotypes. (a) Quantitative RT-PCR of DLC1, ARHGAP5, and double DLC1 and ARHGAP5 KD efficiencies in N1E-115 cells. Mean ± standard deviations from four replicates are shown. (b and c) Quantification of global neurite outgrowth phenotypes of the three perturbations. (b) Representative images of α-tubulin–stained N1E-115 KD cells. Inverted black-and-white contrast. (c) Quantification of total neurite outgrowth. Images were segmented and quantified using the Metamorph neurite outgrowth plugin. The single siRNAs that produced the z-score vector with lowest distance to the mean MDS were used for KD. Population mean ± standard deviations are shown for the whole-cell population (n = 150 cells). One-way ANOVA with Bonferroni’s multiple comparison test was used. ***, P < 0.0001. (d) High-resolution growth cone images of phalloidin-stained, control, and KD growth cones. All experiments were performed and stained simultaneously and acquired with identical acquisition settings. (e) Spatiotemporal RhoA activation patterns in response to the three perturbations. Ratio (RhoA activation), Lifeact-mCherry (F-actin), and overlay representative images of both signals are shown. Cells were cotransfected with the RhoA2G FRET probe, a Lifeact-mCherry construct, and the indicated siRNA. Ratio and F-actin images are color-coded for signal intensity. Ratio images have been scaled identically across experiments. Overlay images highlight the RhoA activity zone (red) over the filopodia F-actin bundles (green). (f) Quantification of RhoA activation in the three perturbed states. The RhoA activity zone was calculated as the pixel percentage of the growth cone RhoA activity area over the growth cone p-domain area. Population mean ± standard deviation are shown for n = 5 cells/sample. One-way ANOVA with Bonferroni's multiple comparison test was used. ***, P < 0.0001. Bars: (b) 100 µm; (d) 10 µm; (e) 10 µm.