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. 2010 Apr 14;30(15):5189–5203. doi: 10.1523/JNEUROSCI.5823-09.2010

Figure 1.

Figure 1.

RNAi knockdown of DMob4 in primary cultures alters neurite morphology. A, Fluorescent microscopy images of Drosophila primary neural cultures derived from embryos expressing GFP. Cultures were either untreated (control) or subjected to DMob4 RNAi using DMob4-specific dsRNA amplicons (DRSC4993 and DRSC29567). B, Quantification of DMob4 RNAi phenotypes in primary neural cultures. Custom algorithms were used to extract and quantify image features from automated microscopy digital files (n = 192 per treatment group). For small neuromeres, a threshold of Loge area ≤5.6 μm2 was set. The number of small neuromeres per image was tabulated as a percentage, and images were binned into groups as displayed in the histogram (bins: 58–67, 67–70, 71–75, or 75–79%). For short neurites, a threshold was set at ≥6.5 and ≤16.1 μm (bins: 63–65, 65–67, 67–68, 68–69, and 69–70%). For long neurites, a threshold was set at ≥25.8 μm (bins: 8.9–9.9, 9.9–10.1, 10.1–12, 12–13, and 13–15%). For neurite strength, representing neurite thickness, bins were set at increments of 31.4 pixel intensity units (PIU). Analysis using all image features indicated that the control is statistically different from treatment groups (control vs DRSC4993, p = 2.22e−16; control vs DRSC29567, p = 1.41e−6), and the two treatment groups are also statistically different from each other (p = 0.002). WT, Wild type. C, Western blot analysis of cell cultures treated with DMob4 dsRNA amplicons. DMob4 protein levels are decreased by ∼75% with DRSC4993 or DRSC29567. No apparent increase in DMob4 knockdown is achieved using a combination of the two amplicons (n = 8). Dlg was used as a loading control. D, DMob4 mRNA in situ hybridization to embryos with DMob4 antisense transcript (blue). DMob4 expression is abundant in early embryos at stage 5 and widely expressed until stage 13, when it becomes enriched in the ventral nerve cord (arrow, stage 14). DMob4 negative control (sense transcript) fails to label the ventral nerve cord [arrow, stage 14, (−) cntrl]. Anterior is to the right, and ventral is down. Scale bars, 100 μm.