Fig. 3.

Effect of CDKL5 expression on proliferation in the SH-SY5Y neuroblastoma cell line.

A,B: Immunofluorescence images of SH-SY5Y neuroblastoma cells transfected with CDKL5-FLAG. Twenty-four hours after transfection, SH-SY5Y cells were treated with BrdU (10 μM) for 2 h and thereafter cells were processed for double immunocytochemistry. Cells were immunostained for FLAG (red signal) and BrdU (green signal in A) or FLAG and Ki-67 (green signal in B). Cell nuclei were stained with Hoechst dye (blue signal). The arrows in Hoechst and merge images indicate the nuclei of cells expressing exogenous CDKL5. Three independent experiments were performed and a minimum of 600 cells were evaluated in each experiment for each condition. Scale bar: 20 μm. C: Labeling index (LI), defined as percentage of BrdU positive cells over total cell number, was determined for SH-SY5Y transfected two different siRNA against CDKL5 (si1 and si2; 50 nM) or with scramble siRNA (siSCR; 50 nM). Forty six hours after transfection cells were exposed to BrdU (10 μM) for the last 2 h. Data, given as percentage of control condition, are expressed as mean ± SE of 3 independent experiments. **P < 0.01; two-tailed t-test.