Fig. 8

NBPF1 overexpression induces nuclear accumulation of p53 as well as p53-dependent cell cycle arrest and CDKN1A induction. (A) HEK293T cells were transfected with a plasmid encoding either EGFP-luciferase (top panels) or EGFP-NBPF1 (lower panels). Immunofluorescence at 48 h after transfection for p53 revealed an increased accumulation in the nucleus upon EGFP-NBPF1 transfection. Nuclei were counterstained with DAPI. Scale bars: 10 μm. (B) HEK293T_shRNAp53 cells were transfected with plasmids encoding either EGFP-luciferase or EGFP-NBPF1. Cell cycle profiles were determined 48 h after transfection by flow cytometry on the effectively transfected (EGFP-positive) cell subpopulations. Transfection efficiency is represented as percentage in the left panels. The different cell cycle phases are shown only for the effectively transfected (EGFP-positive) subpopulations (right panels). Cells in G1 phase (2n DNA content) and cells in G2 phase (4n content) are annotated on the histograms and calculated as percentage of the whole population. NBPF1 overexpression does not induce G1 cell cycle arrest in HEK293T cells with stable knockdown of p53. (C) HEK293T and HEK293T_shRNAp53 cells were transfected with plasmids encoding either EGFP-luciferase or EGFP-NBPF1. Both EGFP-positive (+) and EGFP-negative populations (−) were isolated by FACS, and the expression levels of CDKN1A transcripts were determined by real-time qRT-PCR. EGFP-NBPF1 expression induced CDKN1A transcripts only in HEK293T cells. p-values were calculated with one-way ANOVA; ns: not significant