Allison A. Madigan, Kevin J. Rycyna, Anil V. Parwani, Yeipyeng J

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Novel Nuclear Localization of Fatty Acid Synthase Correlates with Prostate Cancer Aggressiveness Allison A. Madigan, Kevin J. Rycyna, Anil V. Parwani, Yeipyeng J. Datiri, Ahmed M. Basudan, Kathryn M. Sobek, Jessica L. Cummings, Per H. Basse, Dean J. Bacich, Denise S. O'Keefe The American Journal of Pathology Volume 184, Issue 8, Pages 2156-2162 (August 2014) DOI: 10.1016/j.ajpath.2014.04.012 Copyright © 2014 American Society for Investigative Pathology Terms and Conditions

Figure 1 The online program cNLS Mapper indicates the presence of an NLS within the FASN protein sequence (A), immunoblot analysis of LNCaP nuclear lysates indicates that FASN is localized to the nucleus in LNCaP cells (B), and overall expression of FASN is stimulated by testosterone and/or 17β-estradiol (C), but nuclear expression is abolished in the presence of 17β-estradiol. A: The FASN amino acid sequence is shown with the NLS in bold italics and underlined. B: Immunoblot analysis of FASN expression in LNCaP whole cell and nuclear extracts. Levels of nuclear FASN in LNCaPs can vary according to the passage. C: LNCaP cells were treated with 1 nmol/L testosterone, 1 nmol/L 17-β estradiol, or vehicle alone for 4.5 hours. Whole-cell or nuclear extracts were run and anti-FASN, an antibody detecting beta actin, and an antibody detecting the cytoplasmic marker GAPDH are shown. E2, 17-β estradiol; ETOH, ethanol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; T, testosterone; WC, whole-cell extract. The American Journal of Pathology 2014 184, 2156-2162DOI: (10.1016/j.ajpath.2014.04.012) Copyright © 2014 American Society for Investigative Pathology Terms and Conditions

Figure 2 Confocal microscopy confirms nuclear localization of FASN in a subset of prostate cancer cells. FASN (green), Draq5 nuclear stain (blue), and the merge of the two stainings. The American Journal of Pathology 2014 184, 2156-2162DOI: (10.1016/j.ajpath.2014.04.012) Copyright © 2014 American Society for Investigative Pathology Terms and Conditions

Figure 3 Immunohistochemical staining and automated analysis output. Gleason score 3 + 4 = 7 negative control with only secondary antibody staining (A) and the resulting scoring output (E) from the Nuclear Image Analysis algorithm version 9.1 (Aperio). The lines drawn on each section are examples of areas chosen to be anaylzed by the Aperio software. Dashed lines represent areas to be removed from the analysis. A: Red arrows indicate dark nuclei that are still scored as negative because of lack of brown staining. C and D: Yellow arrows indicate specific nuclear localization of FASN. E–H: Blue output coloring from the algorithm indicates no nuclear staining, whereas yellow, orange, and red indicate mild, moderate, and strong nuclear staining, respectively. Gleason pattern 3 + 3 = 6 prostate cancer stained with anti-FASN antibody (B) and scoring output (F), Gleason pattern 3 + 4 = 7 prostate cancer stained with anti-FASN antibody (C) and scoring output (G), and Gleason pattern 4 + 4 = 8 prostate cancer stained with anti-FASN antibody (D) and scoring output (H). The American Journal of Pathology 2014 184, 2156-2162DOI: (10.1016/j.ajpath.2014.04.012) Copyright © 2014 American Society for Investigative Pathology Terms and Conditions

Figure 4 Median and composite nuclear score by pathological group. GS, Gleason score; HGPIN, high-grade prostatic intraepithelial neoplasia. The American Journal of Pathology 2014 184, 2156-2162DOI: (10.1016/j.ajpath.2014.04.012) Copyright © 2014 American Society for Investigative Pathology Terms and Conditions