Fig. 6. Primary cultured neurons (R4HAC) obtained from control and DjCHC-RNAi-treated planarians by FACS. (A) FACS profiles of R4HAC from control body (left), control regenerated head (middle) and regenerated head of the DjCHC-RNAi-treated (right) planarians at 4 days of regeneration. Black ellipses indicate the R4HAC-specific peak of the head region. (B) Percentage (±s.e.m.) of R4HAC among the total cells. (C) Immunostaining with anti-DjCHC (green) and nuclear staining with Hoechst 33342 (blue) of R4HAC of control (upper panels) and DjCHC-RNAi (bottom panels) planarians. The graphs show the percentage (±s.e.m.) of DjCHC protein-expressing cells. (D) Phase-contrast views of R4HAC from control and DjCHC-RNAi-treated planarians. Arrowheads, debris remaining after neurite regression. (E,F) The percentage (±s.e.m.) of neurite-extending cells and the average neurite length per cell of control (white) and DjCHC-RNAi-treated (black) R4HAC. The percentage (±s.e.m.) of neurite-extending cells and average neurite length per cell of DjCHC-RNAi R4HAC compared to those of the control are indicated by the red lines. Data from 200-300 cells from the in vitro analysis were averaged. (G) Immunostaining of R4HAC using anti-DjCHC and DjSYT (green). For counterstaining, planarian 14-3-3 was stained with a specific antibody (red). Nuclei were stained with Hoechst 33342 (blue). ^***P<0.01. NS, not significant; DIV, days in vitro. Scale bars: 10 µm.