KLF4 is a Novel Candidate Tumor Suppressor Gene in Pancreatic Ductal Carcinoma Francesca Zammarchi, Mariangela Morelli, Michele Menicagli, Claudio Di.

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KLF4 is a Novel Candidate Tumor Suppressor Gene in Pancreatic Ductal Carcinoma Francesca Zammarchi, Mariangela Morelli, Michele Menicagli, Claudio Di Cristofano, Katia Zavaglia, Alessandra Paolucci, Daniela Campani, Paolo Aretini, Ugo Boggi, Franco Mosca, Andrea Cavazzana, Luca Cartegni, Generoso Bevilacqua, Chiara Maria Mazzanti The American Journal of Pathology Volume 178, Issue 1, Pages 361-372 (January 2011) DOI: 10.1016/j.ajpath.2010.11.021 Copyright © 2011 American Society for Investigative Pathology Terms and Conditions

Figure 1 A: Example of retention of heterozygosity. Samples are in heterozygosity for the specific microsatellite, and the peaks represent each allele amplification product. Asterisks indicate the peaks chosen for the LOH analysis. LOH analysis was conducted by measuring the ratio between the two peak fluorescence values. LOH was present if the ratio was below 0.5 or above 1.5. B: Example of LOH on D9S105. C: Pattern of LOH on 9q in DPCs. (Black circles are LOH cases; grey circles are cases with homozygous deletions; white circles are cases with retention; white squares are cases with instability; - indicates noninformative cases). The SOR region is highlighted in grey. The American Journal of Pathology 2011 178, 361-372DOI: (10.1016/j.ajpath.2010.11.021) Copyright © 2011 American Society for Investigative Pathology Terms and Conditions

Figure 2 Pattern of LOH on 9q in PanINs (black circles are cases with LOH; grey circles are cases with homozygous deletions; white circles are cases with retention; white squares are cases with instability; — indicates noninformative cases). The SOR region is highlighted in gray. The American Journal of Pathology 2011 178, 361-372DOI: (10.1016/j.ajpath.2010.11.021) Copyright © 2011 American Society for Investigative Pathology Terms and Conditions

Figure 3 A: Schematic representation of RGS3 and KLF4 loci. B: Example of RT-PCR in 5 HomDel and 5 LOH cases. C: Expression of KLF4 in normal pancreatic ducts. D: mRNA expression analysis of the KLF4 gene in HomDel, LOH, ROH, and normal ducts. GAPDH was chosen as an internal control gene. Each histogram represents the ratio of the KLF4 and the GAPDH level of mRNA expression. There is a significant association with a P value = 0.002 between the amount of KLF4 mRNA expression and genomic status. The American Journal of Pathology 2011 178, 361-372DOI: (10.1016/j.ajpath.2010.11.021) Copyright © 2011 American Society for Investigative Pathology Terms and Conditions

Figure 4 A: Five samples of HomDel could not be amplified for any of the genomic coding exons of the KLF4 gene, whereas all LOH and ROH samples were successfully amplified (figure shows only a few representative samples). B: The Braf gene was successfully amplified in all five HomDel samples and in a few representative LOH and ROH samples as a control for the genomic DNA used to support the result of a KLF4 locus homozygous deletion. The American Journal of Pathology 2011 178, 361-372DOI: (10.1016/j.ajpath.2010.11.021) Copyright © 2011 American Society for Investigative Pathology Terms and Conditions

Figure 5 KLF4 promoter methylation analysis. A: A fragment of KLF4 5′ UTR showing orginal nucleotide sequence before bisulfite treatment; cytosines of 3 CpG dinucleotides are circled with an asterisk. B: Electropherogram of an unmethylated case. C: Electropherogram of a methylated case. The American Journal of Pathology 2011 178, 361-372DOI: (10.1016/j.ajpath.2010.11.021) Copyright © 2011 American Society for Investigative Pathology Terms and Conditions

Figure 6 Immunostaining with anti-KLF4 antibody: negative (A) and positive (B) PanINs; negative (C) and positive (D) DPCs (400× magnification). The American Journal of Pathology 2011 178, 361-372DOI: (10.1016/j.ajpath.2010.11.021) Copyright © 2011 American Society for Investigative Pathology Terms and Conditions

Figure 7 Effect of KLF4 on cell proliferation and viability. The following assays were performed on PANC-1 cells 72 hours after transfection with either KLF4 or the empty-vector. A–B: Cell proliferation was assayed by either cell count or the MTT proliferation assay; columns represent mean of two independent experiments done in triplicate; bars, SD. C: Western blot analysis checked the expression level of KLF4, p21 and cyclin D1. Tubulin was used as loading control. D: Cell death was determined by trypan blue assay. Values are presented as dead/alive percent; columns represent the mean of two independent experiments done in triplicate; bars, SE *P <0.05. The American Journal of Pathology 2011 178, 361-372DOI: (10.1016/j.ajpath.2010.11.021) Copyright © 2011 American Society for Investigative Pathology Terms and Conditions

Supplemental figure 1: A) MTT proliferation assay on PANC-1 after 96h from transfection with either KLF4 or the empty vector. columns represent mean of two independent experiments done in triplicate; bars, SD. B) BxPC3 cells were transfected with either KLF4 or the empty vector. After 72h from transfection cell proliferation was assayed be cell count. columns represent mean of two independent experiments done in triplicate; bars, SD. * p-value <0.05. The American Journal of Pathology 2011 178, 361-372DOI: (10.1016/j.ajpath.2010.11.021) Copyright © 2011 American Society for Investigative Pathology Terms and Conditions