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Fig. 8. P-Rex1-depleted neurites exhibit small growth cones with decreased F-actin. (A) Embryonic rat hippocampal cells (1 d.i.v.) were transiently transfected with plasmids encoding P-Rex1 RNAi or a control RNAi. Cells were co-transfected with a low concentration of pEGFP-C2 vector (ratio of 0.5 µg eGFP:1.5 µg RNAi plasmid) to allow for identification of RNAi P-Rex1-depleted neurons. At 3 d.i.v., cells were stained with Texas Red-conjugated phalloidin to visualise F-actin. Representative images are shown in the left column, with magnified images of the boxed growth cones (1-4) shown in the middle and right-hand columns. Scale bars: 10 µm. (B-D) The indicated scrambled RNAi and P-Rex1 PC12 RNAi clones were NGF-differentiated for 3 days, co-stained with Alexa Fluor 488-conjugated phalloidin to visualise F-actin and Alexa Fluor 594-conjugated DNaseI to visualise G-actin and analysed using confocal microscopy. (B) Representative images of growth cone F-actin. Lamellipodial veils are indicated by arrows. Scale bar: 5 µm. (C) The mean fluorescence intensity of F-actin relative to G-actin at the growth cone was determined. Bars indicate the mean ± s.e.m. of the F-actin:G-actin ratio for at least ten growth cones per RNAi clone for each of three independent differentiation experiments. (D) Growth clones were scored for the presence of lamellipodial veils by F-actin morphology. Bars indicate the mean ± s.e.m. for the percentage of cells containing growth cone lamellipodial veils for at least ten growth cones per RNAi clone for each of three independent differentiation experiments. *P<0.05.
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