Figure 1

HPV-16 E7 induces centriole overduplication through transcriptional deregulation and aberrant recruitment of PLK4 to maternal centrioles. (A) Quantification of centriole overduplication (>4 centrioles per cell) in U-2 OS/centrin-GFP cells transfected with either control (siControl) or PLK4 (siPLK4) siRNA duplexes for 24 h followed by ectopic expression of HPV-16 E7 for another 48 h. Mean and standard error of at least three independent experiments with at least 100 cells counted per experiment are shown. Asterisk indicates statistically significant differences (p ≤ 0.002). (B) Immunofluorescence microscopic analysis of human keratinocytes transduced with either empty vector (LXSN) or HPV-16 E7 for endogenous PLK4 following transiently transfected with centrin-GFP (48 h). Arrows indicate PLK4 dots at maternal centrioles. Note the presence of two dots in a HPV-16 E7-expressing cell (bottom panels). (C) Quantification of the percentage of cells with aberrant (two or more) PLK4 dots at maternal centrioles in human keratinocytes transduced with either a control plasmid, LXSN, or HPV-16 E7. Asterisk indicates statistically significant differences (p ≤ 0.05). (D) Quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) analysis for PLK4 mRNA was performed on total RNA isolated from human keratinocytes expressing either a control plasmid, LXSN, or HPV-16 E7. βactin was used as a control housekeeping gene. Experiments were performed in triplicate and analyzed as described in the Materials and Methods section (Additional File 1). Asterisks indicate statistically significant differences (p ≤ 0.01). (E) Fluorescence microscopic analysis of U-2 OS/centrin-GFP cells transfected with either an empty vector control plasmid (pCMV-Neo) or E2F-1 for 48 h. Nuclei stained with DAPI. Scale bar indicates 10 μm. (F) Quantification of centriole multiplication (>4 centrioles, >1 daughter at one or more maternal centrioles) in U-2 OS/centrin-GFP cells ectopically expressing either a control plasmid or E2F-1 for 48 h. Mean and standard error of two independent experiments with a triplicate of at least 100 cells counted per experiment are shown. Asterisk indicates statistically significant differences (p ≤ 0.002). (G) Quantification of PLK4 promoter activity following transient co-transfection (48 h) of U 2-OS/centrin-GFP cells with the PLK4 promoter construct (pGL3-PLK4) and a transfection control (pRL-CMV) with either an empty vector control (Control) or E2F-1. The bar graph shows fold activation of the PLK4 promoter when compared with the empty luciferase construct, pGL-3, from three independent transfections, and expressed as mean and standard error. Asterisks indicate statistically significant differences (p ≤ 0.001). (H) Table comparing quantification of centriole overduplication (>4 centrioles), PLK4 promoter activation, and PLK4 mRNA upregulation in U-2 OS/centrin-GFP cells ectopically expressing the indicated construct for 48 h and analyzed as described in the Materials and Methods section (Additional File 1).