shRNA-Mediated Silencing of Y-Box Binding Protein-1 (YB-1) Suppresses Growth of Neuroblastoma Cell SH-SY5Y In Vitro and In Vivo
Fig 3
YB-1 regulated Cyclin D1 transcription in neuroblastoma SH-SY5Y cells.
(A) Expression levels of cell cycle regulators such as Cyclin A and Cyclin D1 in SH-SY5Y, SH-shCON and SH-shYB-1 cells were examined by Western blot analysis. (B) A schematic illustration of the promoter region of CCND1 which encodes Cyclin D1 indicates the transcription start site (TSS), putative YB-1 binding site and the location of two sets of primers (CCND1p #1 and #2) used for chromatin-immunoprecipitation (ChIP) assay. (C) ChIP assay followed by PCR analysis was performed in SH-SY5Y cells to detect binding of YB-1 on the promoter region of CCND1. Binding of RNA polymerase II (RNA PolII) on GAPDH promoter, which was detected with primers for GAPDH promoter (GAPDHp) by PCR, was used as a positive control for ChIP experiments, whereas IgG served as a negative control for non-specific binding. (D) pGL-3 Firefly luciferase reporter plasmid containing a CCND1 promoter fragment (pGL-3-CCND1) or pGL-3 vector alone was transfected in combination with Renilla luciferase reporter vector pRL-TK into SH-SY5Y, SH-shCON and SH-shYB-1 cells. Luciferase activity representing activity of the promoter was quantified as the ratio of FL/RL which was then normalized to SH-SY5Y cells. Values are expressed as mean ± standard deviation. Compared with SH-SY5Y control, ***P<0.001.