Fig 5.

NF45 regulates survivin and cyclin E expression downstream of XIAP and cIAP1. (A) Immunofluorescence images showing the multinucleation phenotype of the d5 HeLa cell line (arrow, bottom left panel) compared to the c HeLa cell line. The same phenotype can be reproduced by the transient siRNA knockdown of NF45 for 72 h (arrow, bottom right panel and inset). Cells were stained with Alexa Fluor 568 phalloidin for F-actin and with Hoechst stain for nuclei. (B) c and d5 cell phenotypes were quantified by propidium iodide staining and flow cytometry analysis. The number of multinucleated cells expressed as a percentage of the total number of viable cells was quantified and normalized to that of the control cell line (bottom). The number of cells in G₂/M phase is also shown. (C) Western blot and densitometry analyses showing increased survivin expression in HeLa cells treated with 50 nM NF45 siRNA for 96 h compared to control siRNA. (D) survivin expression was blunted by XIAP overexpression in NF45 knocked-down HeLa cells. The HeLa cells were transfected with 50 nM NF45 or control siRNA and transfected 48 h later with GFP-XIAP for an additional 48 h. The protein extracts were analyzed by Western blotting and densitometry for survivin, XIAP, NF45, and GAPDH. (E) NF45 regulates cyclin E expression. HeLa cells were treated with 50 nM NF45 or control siRNA for 96 h, and expression of the indicated proteins was analyzed by Western blotting and densitometry. (F) NF45 controls cyclin E protein levels through its regulation of cIAP1 IRES-mediated translation. The HeLa cells were treated with 100 nM Smac mimetic (SM) or DMSO for 24 h and transfected with a pcDNA3-GFP or pcDNA3-GFP-NF45R plasmid for an additional 24 h. Protein extracts were analyzed by Western blotting for NF45, cIAP1, cyclin E, and GAPDH expression, and densitometry was performed.