Supplementary Figure 5: Bmp2 proximal promoter is preferentially used in neurospheres.

(a) Schematic representation of the murine Bmp2 promoter (grey bar) showing two alternative transcription initiation sites (TSS) at +1 and −736, and the two fragments used for reporter experiments: distal, (−2712/−700)-luciferase, red bar, and proximal, (−643/+165)-luciferase, purple bar. Neurospheres were transfected with either of these reporter constructs and luciferase activity was measured 24 h later. (b) Interfered shRNAp21-c17.2 cells were transfected with an E2F-luciferase reporter bearing 3 tandem repeats of the hydropholate reductase E2F-binding site (E2F-luc) and treated with 2.5 μM CDK inhibitor 2-bromo-12,13-dihydro-5H-indolo [2,3-a] pyrrolo [3,4-c] carbazole-5,7(6H)-dione. Interfered shRNAp21-c17.2 cells were transfected in parallel with the proximal Bmp2 promoter in the presence or in the absence of the inhibitor. (c) p21-null neurospheres were transfected with the E2F-luciferase reporter (E2F-luc), or co-transfected with pCDNA-p21 and the proximal Bmp2-promoter, and cells were treated or not with 40 μM E2F inhibitor HLM006474. Luciferase activity was measured 24h after transfection. (d) Immunoblot for Flag epitope showing the expression of the p21-mutants used (upper panel) and co-immunoprecipitation of p21-Flag and CDK2 or Cyclin A in mouse fibroblasts transfected with the different p21-mutants used (bottom panels). (e) Percentage of cells transfected with the different p21-mutant constructs that are retained in G1 phase of the cell cycle shown as increase relative to empty vector values. Data are shown as mean values ± s.e.m. (n = 4, panels a–c; n =2, panel e); **p<0.05,**p<0.01, **p<0.001.