Fig. 5

c-Myc is a transcriptional regulator of UHFR1 which promotes stemness and invasion of GBM human GSCs. (A) Western blots to confirm siRNA knockdown efficiency of UHRF1 in P3 and BG5 GSCs. (B) Extreme limiting dilution assay performed with P3 and BG5 human GSCs transfected with si-NC, si-UHRF1-2 or si-UHRF1-3. Data are shown as the mean ± SEM. Statistical significance was determined by ANOVA. (C) Quantification of the tumorsphere formation assays for P3 and BG5 GSCs transfected with si-NC, si-UHRF1-2 or si-UHRF1-3. Data are shown as the mean ± SEM. Statistical significance was determined by ANOVA. ***P < 0.001. (D) Quantification of the invasion in the 3D invasion spheroids assays for P3 and BG5 transfected with si-NC, si-UHRF1-2 or si-UHRF1-3. All data are presented as the mean ± SEM, Statistical significance was determined by ANOVA. ***P < 0.001. (E) Western blots to confirm siRNA knockdown efficiency of c-Myc in P3 and BG5 human GSCs. (F and G) Representative images and statistical analysis from tumorsphere formation assays for P3 and BG5-c-Myc-OE/-NC human GSCs (overexpression of c-Myc or control) treated with SKF83566. Scale bar = 200 µm. Data are shown as the mean ± SEM. Statistical significance was determined by the unpaired Student’s t-test. *P < 0.05, **P < 0.01 and ***P < 0.001. (H) Statistical analysis from 3D invasion spheroids assays for P3 and BG5-c-Myc-OE/-NC treated with SKF83566. Data are shown as the mean ± SEM Statistical significance was determined by unpaired Student’s t-test. **P < 0.01, ***P < 0.001. (I) Predicted c-Myc transcriptional binding sites from the JASPER database. (J) Quantification of the ChIP-PCR assay performed with antibodies against c-Myc to detect c-Myc binding to the UHRF1 promoter. Input was used for normalization and IgG was used for negative control. Data are shown as the mean ± SEM. Statistical significance was determined by unpaired Student’s t-test. **P < 0.01. (K) Construction of wild type (WT) and mutant (MUT) luciferase reporter vectors based on the predicted binding site of c-Myc in the 3’ UTR of UHRF1. (L) Quantification of luciferase activity from HEK293t cells co-transfected with the reporter vectors and c-Myc-OE or c-Myc-NC. Luciferase activity was assessed 48 h after transfection. Data are shown as the mean ± SEM. Statistical significance was determined by the unpaired Student’s t-test. ****P < 0.0001