Figure 4
From: The HIF1α/JMY pathway promotes glioblastoma stem-like cell invasiveness after irradiation
The HIF1α/JMY pathway is involved in radiation-induced migration of GSCs. (A) JMY immunostaining (white) of GSCs 24 h after 0 Gy or 0.5 Gy irradiation. F-actin filament networks are stained with fluorescent-phalloidin (red). Nuclei were counterstained with DAPI (blue). Scale bar: 20 µm. (B) Quantification of JMY fluorescence intensity. At least 20 cells were scored per condition. (***p < 0.001). (C) Quantification of JMY mRNA levels by RT-qPCR in TG1N cells. Experiments were performed in triplicate (*p < 0.05, **p < 0.01 and ***p < 0.001). (D) JMY promoter activity in TG1N cells was estimated by luciferase reporter assay at different times following irradiation (0.5 Gy). Data obtained from quadruplicates (*p < 0.05, **p < 0.01 and ***p < 0.001). (E) TG1N cells were treated with 50 µM YC1 one hour before irradiation (0.5 Gy). Immunostainings were performed 24 h after irradiation or after treatment with 100 µM DFO. JMY-fluorescence intensity was measured (n = 30 cells per condition; ***p < 0.001). (F) Quantification of JMY mRNA levels by RT-qPCR in control TG1N cells (shCt) and in HIF1α-deficient TG1N cells. Experiments were performed in duplicate (*p < 0.05). (G,H) Twenty-four hours after irradiation (0.5 Gy), cells transfected with siCt or siJMY (F) or transduced with shJMY-TG1N or shCt-TG1N cells (G) were tracked every 10 min for 4 h. Data were obtained from at least 65 cells per condition (***p < 0.001). The results are expressed as percentages of unirradiated controls (siCt TG1N cells (F) or shCt TG1N cells (G)). (I) Invasion in Matrigel chambers of ShJMY-TG1N cells and ShCt-TG1N cells after 0.5 Gy radiation. GFP-positive cells present on the lower membrane were numbered. For each condition, results were expressed as percentages of unirradiated ShCt-TGIN cells (n = 3 chambers per group; **p < 0.01). (J) Quantification of the dispersion of unirradiated or irradiated ShJMY-TG1N cells and ShCt-TG1N cells in coronal slices of nude mouse brains 48 h after intrastriatal injections (n = 3–5 brains per condition; *p < 0.05).
