Fig. 5
PBX3 activates LIN28/let-7b axis via ERK1/2 and c-myc-dependent mechanisms. a GBM cells stably expressing LV-PBX3 or LV-NC were treated with U0126 for 24 h and then LIN28 protein levels and let-7b expression were determined by western blot and qRT-PCR assays, respectively. **indicates a statistical significant difference (p < 0.01) between LV-NC + U0126 (−) group and LV-NC + U0126 (+) group. # indicates a statistical significant difference (p < 0.01) between LV-NC + U0126 (−) group and LV-PBX3 + U0126 (−). $ indicates a statistical significant difference (p < 0.01) between LV-PBX3 + U0126 (−) group and LV-PBX3 + U0126 (+) group. b GBM cells stably expressing LV-siRNA-PBX3 or LV-siRNA-NC were treated with PMA for 24 h and then LIN28 protein levels and let-7b expression were determined by western blot and qRT-PCR assays, respectively. **indicates a statistical significant difference (p < 0.01) between LV-siRNA-NC + PMA (−) group and LV-siRNA-NC + PMA (+) group. # indicates a statistical significant difference (p < 0.01) between LV-siRNA-NC + PMA (−) group and LV-siRNA-PBX3 + PMA (−). $ indicates a statistical significant difference (p < 0.01) between LV-siRNA-PBX3 + PMA (−) group and LV-siRNA-PBX3 + PMA (+) group. c GBM cells infected with c-myc overexpressing plasmids or empty vectors were treated with U0126 for 24 h and then LIN28 and c-myc protein levels were determined by western blot assays. Let-7b expression were measured by qRT-PCR assays. **indicates a statistical significant difference (p < 0.01) between vector + U0126 (−) group and vector + U0126 (+) group. # indicates a statistical significant difference (p < 0.01) between vector + U0126 (−) group and c-myc + U0126 (−). $ indicates a statistical significant difference (p < 0.01) between c-myc + U0126 (−) group and c-myc + U0126 (+) group. d GBM cells infected with siRNA-NC or siRNA-c-myc were treated with PMA for 24 h and then LIN28 and c-myc protein levels were determined by western blot assays. Let-7b expression were measured by qRT-PCR assays. **indicates a statistical significant difference (p < 0.01) between siRNA-NC + PMA (−) group and siRNA-NC + PMA (+) group. # indicates a statistical significant difference (p < 0.01) between siRNA-NC + PMA (−) group and siRNA-c-myc + PMA (−). $ indicates a statistical significant difference (p < 0.01) between siRNA-c-myc + PMA (−) group and siRNA-c-myc + PMA (+) group. e c-myc regulates LIN28 expression by binding to its promoter in GBM cells. Diagram of amplicons for ChIP-qPCR were illustrated. ChIPs were performed with anti-c-myc antibody and anti-Jun antibody (a positive control). ChIP was analyzed by qRT-PCR, with primers in the LIN28, WNT5A (a positive control), CyclinD1 (a positive control), and β-globin (a negative control) promoters. f U0126 treatment reversed the effect of PBX3 overexpression-induced enhanced c-myc binding to LIN28 promoters. **indicates a statistical significant difference (p < 0.01) between LV-NC + U0126 (−) group and LV-PBX3 + U0126 (+) group. # indicates a statistical significant difference (p < 0.01) between LV-NC + U0126 (−) group and LV-NC + U0126 (+). $ indicates a statistical significant difference (p < 0.01) between LV-PBX3 + U0126 (−) group and LV-PBX3 + U0126 (+) group. g PMA treatment reversed the effect of PBX3 knockdown-induced decreased c-myc binding to LIN28 promoters. **indicates a statistical significant difference (p < 0.01) between LV-siRNA-NC + PMA (−) group and LV-siRNA-PBX3 + PMA (+) group. # indicates a statistical significant difference (p < 0.01) between LV-siRNA-NC + PMA (−) group and LV-siRNA-NC + PMA (+). $ indicates a statistical significant difference (p < 0.01) between LV-siRNA-PBX3 + PMA (−) group and LV-siRNA-PBX3 + PMA (+) group