Figure 4. Knockdown of Espn disrupts F-actin localization and dendritic development in PCs.

(a) Semiquantitative RT-PCR showing significant downregulation of Espn in cerebellar slice cultures after transfection of Espn siRNA; n=3 mice per group. (b) Western blots showing significant knockdown of Espin protein in stable HEK293A cells expressing Espin-DsRed after transfection with Espn siRNA; n=3 per group. (c) Representative PCs from P14 cerebellum slice cultures co-transfected with pDsRed and nontargeting siRNA (left panels) or Espn siRNA (right panels) and collected at 3 days in vitro (DIV). Zoom-in images of yellow boxed regions show the dendritic spines of the respective PCs. White arrows point to the mature spines while arrowheads point to the immature spines. Scale bars, 20 μm; 10 μm (zoom-in). (d) Bar graphs showing the total dendritic length (left) and the branching number (right) of PCs were significantly reduced after transfection of Espn siRNA. (e) Bar graphs showing the density of dendritic spines (left) and the percentage of mature spines (right) of PCs were significantly reduced after transfection of Espn siRNA. Brackets show the number of transfected PCs analysed. N=7–8 mice; t-test; ***P<0.001. (f,g) F-actin cytoskeleton was visualized by co-transfection of EGFP-Actin into P14 PCs transfected with nontargeting siRNA (f) or Espn siRNA (g). Only the central focal planes of the PC proximal dendrites were shown here. Scale bars, 10 μm. Fluorescence intensity profile plots (across the yellow doted lines) reveal the distribution of F-actin in the dendritic shafts of the proximal dendrites of the transfected PCs, respectively. Areas shaded in cyan represent the dendritic shafts. Each profile plot was sampled from 11 different PCs from 4 to 6 different mice and the curves represent the mean values with surrounding shaded regions as s.e.m. Note the shift of F-actin to the centre of the dendritic shafts in the PCs transfected with Espn siRNA.