Fig. 3

DRD1 is the target of SKF83566 in human GSCs. (A) SwissTargetPrediction of the interaction of SKF83566 with the predicted target DRD1. (B) Western blots to detect protein levels of DRD1 in NHA, three GBM cell lines (A172, LN229, and U251), and three human GSCs (P3, BG5 and BG7). (C) Western blot to confirm siRNA knockdown efficiency of DRD1 in P3 and BG5 human GSCs. (D) Extreme limiting dilution assay performed with P3, BG5 human GSCs transfected with si-DRD1. (E) Quantification of the tumorsphere formation assays for P3 and BG5 GSCs transfected with si-NC, si-DRD1-2 or si-DRD1-3. Data are shown as the mean ± SEM. Statistical significance was determined by ANOVA. ***P < 0.001. (F and G) Representative images and quantification of the co-culture invasion assays for P3 and BG5 human GSCs transfected with si-NC, si-DRD1-2 or si-DRD1-3. Scale bar = 100 μm. Data are shown as the mean ± SEM. Statistical significance was determined by ANOVA. ***P < 0.001. (H and I) Representative images from tumorsphere formation assays for P3 and BG5 human GSCs through overexpression of DRD1 followed by SKF83566 treatment. 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. (J and K) Representative images of spheroids in the 3D invasion assays for P3- and BG5-DRD1-OE/-NC (overexpression of DRD1 or control) followed by SKF83566 treatment, and evaluated at 96 h. Scale bar = 200 μm. Quantification of the distance of invading cells from the tumorspheres determined after 96 h. Data are shown as the mean ± SEM. Statistical significance was determined by the unpaired Student’s t-test. **P < 0.01 and ***P < 0.001