Figure 3: FHL1 sequesters CDC25C in the cytoplasm via 14-3-3ɛ.

(a) HeLa or MCF-7 cells stably infected with FHL1 shRNA or control shRNA were exposed to IR, fractionated into cytoplasmic (Cyto) and nuclear (Nuc) fractions and analysed by immunoblot with the indicated antibodies. (b) HeLa or MCF-7 cells stably infected with FHL1 shRNA or control shRNA were transfected with 14-3-3ɛ siRNAs, exposed to IR, fractionated into cytoplasmic and nuclear fractions, and analysed by immunoblot with the indicated antibodies. C, cytoplasm; N, nucleus. Right panels show the densitometric quantitation of cytoplasmic and nuclear distribution of CDC25C (a,b). Values shown are mean±s.d. of three independent experiments. The P values were generated using two-tailed Student’s t-test. ^#P<0.05 versus control shRNA without IR (the ratio of nucleus to cytoplasm), **P<0.01 versus control shRNA with IR (a,b). (c) HeLa cells transfected with the indicated plasmids were IP with anti-FLAG. The immune complexes were eluted with FLAG peptide, Re-IP using anti-Myc and analysed by immunoblotting with the indicated antibodies. (d) HeLa cells stably infected with FHL1 shRNA or control shRNA were irradiated and IP with anti-14-3-3ɛ or normal IgG. The resulting precipitates were analysed by immunoblotting with the indicated antibodies. (e) Representative immunohistochemical staining of FHL1 and CDC25C in human breast cancer samples. Case 1 and case 2 refer to two representative samples categorized by low and high expression of FHL1 and nuclear and cytoplasmic expression of CDC25C. Scale bars, 100 μm. The correlation of FHL1 with nuclear or cytoplasmic CDC25C is shown. The correlation of FHL1 with nuclear or cytoplasmic CDC25C was determined using the Mann–Whitney U test. Nuclear and cytoplasmic expression scores are shown in box plots, with the horizontal lines representing the median; the bottom and top of the boxes representing the 25th and 75th confidence limits; and the vertical bars representing the range of data.