Fig. 4.

Expression of CDKL5 does not induce cell death but blocks cell cycle progression in SH-SY5Y neuroblastoma cells.

A: Example of immunofluorescence image of SH-SY5Y neuroblastoma cells transfected with GFP-CDKL5. Twenty-four hours after transfection, SH-SY5Y cells were immunostained for cleaved caspase-3 (red signal). Cell nuclei were stained with Hoechst dye (blue signal). Scale bar: 25 μm. B: Percentage of cleaved caspase-3-positive cells over total number of GFP-CDKL5-positive cells and GFP-positive cells. Data are expressed as mean ± SE of 3 independent experiments. A minimum of 450 cells were evaluated in each experiment for each condition. C: Twenty-four hours after transfection with GFP-CDKL5 or GFP alone, SH-SY5Y cells were FACS sorted for GFP expression. The threshold for positivity for GFP was established as the levels corresponding to untransfected cells (histogram on the left). The green area in the histogram on the right shows fluorescence intensity of sorted GFP-CDKL5 positive cells. D: Distribution of cell populations in the G_0/1, S, or G₂/M phases of the cell cycle, identified by flow cytometry analysis. Data, given as percentage of either GFP-CDKL5 or GFP cells in each phase of the cell cycle, are expressed as mean ± SE of 3 independent experiments, *P < 0.05, **P < 0.01, two-tailed t-test.