Fig. 5.

P27 knockdown inhibits neuronal differentiation and increases EdU‐positive cells of SH‐SY5Y cells. SH‐SY5Y cells were infected with sh‐P27 or control (sh‐Neg) lentiviruses. (A) Western blot analysis of the indicated protein expression in noninfected, control virus (sh‐neg), or sh‐P27 lentiviruses infected SH‐SY5Y cells, which were cultured in the presence of 10 μm RA for 4 days. (B) Western blot analysis of MAP2 protein expression in control (sh‐Neg), or sh‐P27 lentiviruses infected SH‐SY5Y cells, which were cultured in the absence or presence of RA for 8 days. (C) Immunostaining of neuronally differentiated SH‐SY5Y cells, which were cultured in the presence of 10 μm RA for 8 days, with an anti‐MAP2 antibody. Representative photomicrograph of MAP2‐positive cells. The neurite length was calculated in differentiated SH‐SY5Y cells (arrows: representative neurite). (D) SH‐SY5Y cells were aggregated in the presence of 10 μm RA for 4 days and incubated with EdU for 2 h before fixation. EdU‐positive cells were used for immunofluorescence analysis. The percentage of EdU‐positive cells was determined. Scale bars, 200 μm. Data are depicted as means ± SD of at least three independent experiments. **P < 0.01, and ***P < 0.001, as determined by the two‐tailed unpaired Student's t‐test (C) and one‐way ANOVA followed by the LSD post hoc test (D).