Fig. 1
CXCL12 activates AurA through CXCR4 and ERK1/2 proteins in glioblastoma cells. a Immunofluorescent staining and normalized percentage of P-AurA and AurA/Hoechst double-positive U87MG and GBM1 cells after 0, 1, 4, 8, 16, 24, 48 h of CXCL12 stimulation (12.5 nM). Pictures show P-AurA (red), AurA (green), and Hoechst (blue) staining of U87MG stimulated during 16 h with 0 or 12.5 nM CXCL12. The arrows show P-AurA/AurA double-positive cells (40×). Scale bars = 10 μm, (n = 3). b Western-blot analysis of P-AurA, AurA, and GAPDH in U87MG and GBM1 cells control (CT) or stimulated during 16 h with CXCL12 (12.5 nM) (n = 3). c AurA kinase activity assay on U87MG and GBM1 cells upon 16 h of stimulation with CXCL12 (12.5 nM). Graphs show quantified luminescence (in Relative Luminescence Units, RLU) plotted semi-logarithmically against the of AurA immunoprecipitate amount (n = 3). d Normalized percentage of P-AurA and AurA/Hoechst double-positive U87MG cells immunofluorescent staining after CXCL12 stimulation (16 h, 12.5 nM) (n = 3). U87MG X4/X7− are negative for CXCR4 and CXCR7, U87MG X4+ overexpress CXCR4, and U87MG X7+ overexpress CXCR7. e Normalized percentage of P-AurA and AurA/Hoechst double-positive U87MG and GBM1 cells from immunofluorescent staining after CXCL12 stimulation (16 h, 12.5 nM). U87MG and GBM1 cells were treated with AMD3100 (24 h, 25 nM), U0126 (24 h, 10 μM), and Alisertib (48 h, 5 nM) treatments (n = 3). f Western-blot analysis of ERK1/2 and GAPDH in U87MG cells control (un-transfected) or transfected with siRNA CT (scramble control), siERK1 A, and siERK1 B. g Normalized percentage of P-AurA and AurA/Hoechst double-positive U87MG and GBM1 cells from immunofluorescent staining after CXCL12 stimulation (16 h, 5 nM). U87MG cells were transfected with siRNA CT (scramble control), siERK1 A, and siERK1 B (n = 3). h Quantitative RT-PCR analysis of Ajuba and TPX2 in U87MG cells after CXCL12 and/or CXCL12 treatments (n = 3). Graphs are mean values ± SD and are representative of three independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001 (t-tests, 1-way ANOVA, 2-way ANOVA corrected by post-tests if appropriate)
