Fig. 1

Effect of P021 treatment on cell proliferation, survival, and differentiation in SH-CDKL5-KO cells. A Schematic representation of the experimental design to assess proliferation and survival in SH-SY5Y and SH-CDKL5-KO cells. B Representative fluorescence images of Hoechst-stained nuclei (white arrows indicate pyknotic nuclei, yellow arrows indicate mitotic nuclei) of untreated and P021 (10 µM)-treated SH-SY5Y (SH) and SH-CDKL5-KO cells. C Percentage of mitotic cells in untreated (0 µM) and P021 (1 µM and 10 µM)-treated SH-SY5Y and SH-CDKL5-KO cells 24 h after the treatment administration. D Percentage of pyknotic nuclei in untreated and P021 (10 µM)-treated SH-SY5Y and SH-CDKL5-KO cells. E Percentage of mitotic cells in SH-SY5Y and SH-CDKL5-KO cells, expressed as ratio of P021-treated to untreated cells. F Percentage of pyknotic nuclei in SH-SY5Y and SH-CDKL5-KO cells, expressed as ratio of P021-treated to untreated cells. G Schematic representation of the experimental design to assess differentiation in SH-SY5Y and SH-CDKL5-KO cells. Cells were treated daily with retinoic acid (RA; 10 μM) and P021 (10 μM) for 5 days. H Quantification of neurite outgrowth after RA-induced differentiation with or without P021 treatment, in parental cell line (SH) and SH-CDKL5-KO cells, expressed as a percentage of the SH-SY5Y untreated condition. I Representative phase-contrast images of neurite outgrowth of RA-treated and RA/P021-treated SH-SY5Y (SH) and SH-CDKL5-KO cells. J Western blot analysis of phospho-GSK3β (Ser9; P-GSK3β) levels in protein extracts from untreated (SH n = 5; SH-CDKL5-KO n = 5) and P021-treated SH-SY5Y (SH n = 3) and SH-CDKL5-KO (n = 4) cells. The histogram shows P-GSK3β protein levels normalized to total GSK3β levels. Data are expressed as a percentage of vehicle-treated SH-SY5Y cells. Examples of immunoblot for P-GSK3β, GSK3β, and GAPDH in the lower panel. Values are represented as means ± SEM of 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001. Fisher’s LSD test after two-way ANOVA for data set in (C, D, H, J) and Student’s t-test for data set in (E, F)