Plakophilin-1 Localizes to the Nucleus and Interacts with Single-Stranded DNA  Tammy Sobolik-Delmaire, Roopa Reddy, Anjeza Pashaj, Brett J. Roberts, James K. Wahl  Journal of Investigative Dermatology  Volume 130, Issue 11, Pages 2638-2646 (November 2010) DOI: 10.1038/jid.2010.191 Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Plakophilin (Pkp) expression and subcellular localization. A431 cells expressing Pkp-1 (a, e, i, and k), A431 (c, d, g, and h), and UM-SCC-38 (b, f, j, and l) cells were stained using antibodies specific for Pkp-1 (14B11; a–c and 19F10; e–g), Pkp-2 (8H6; i and j), and Pkp-3 (19A5; k and l). Panels d and h show 4,6-diamidino-2-phenylindole staining of cells in panels c and g. Pkp-1 is prominently localized to nuclei, whereas Pkps-2 and -3 are mainly localized at cell–cell borders. (m) Total cell lysates were prepared from A431/Pkp-1 and UM-SCC-38 cells, equal protein was resolved by SDS-PAGE, and membranes were immunoblotted with the indicated antibodies. (Bar in panel a=10μm.) Journal of Investigative Dermatology 2010 130, 2638-2646DOI: (10.1038/jid.2010.191) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Subcellular localization of plakophilin (Pkp) chimeras. A431 cells expressing Pkp-1 (a and b), Pkp-13 (c and d), and Pkp-31 (e and f) were stained with antibodies specific for the amino-terminal head domain of Pkp-1 (a and c) or the armadillo repeat domain of Pkp-1 (b and d). (Bar in panel d=10μm.) Journal of Investigative Dermatology 2010 130, 2638-2646DOI: (10.1038/jid.2010.191) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Nuclear localization of plakophilin-1 (Pkp-1) deletion mutants. A431 cells expressing Pkp-1 AA 1–235 (a–d), Pkp-1 AA 235–726 (e–h), Pkp-1 AA 56–726 (i–l), and Pkp-1 AA 126–726 (m–p) were immunostained using the appropriate anti-Pkp-1 antibodies without Triton X-100 extraction (left panels) or after extraction in 0.2% Triton X-100 (right panels). (Bar in panel p=10μm.) Journal of Investigative Dermatology 2010 130, 2638-2646DOI: (10.1038/jid.2010.191) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Plakophilin-1 (Pkp-1) binds DNA. Pkp-1 is released from the nucleus by DNase I. A431/Pkp-1 cells were untreated (a and b) or treated with DNase I (c and d) before fixation and stained using anti-Pkp-1 (a and c) or visualized using 4,6-diamidino-2-phenylindole (b and d). Note the longer exposure in panel c compared with panel a to visualize the small amount of Pkp-1 remaining after DNaseI treatment (bar in panel c=10μm). (e) Recombinant glutathione S-transferase (GST)-Pkp-1 (AA 1–235), GST-Pkp-2 (AA 1–360), GST-Nck-1 (AA 250–377), GST alone, and replication protein A (RPA) were applied to a single-stranded DNA cellulose column and eluted using increasing NaCl as indicated. (f) Recombinant protein was added to microtitre plates (three wells for each fusion protein) coated with oligo-dG and bound fusion protein was detected using anti-GST. Each assay was performed in triplicate wells of a microtitre plate and a representative assay is presented. Error bars represent the SD of one triplicate assay. Analysis of variance was used to determine that each group shown in panel f was statistically different from the others (P<0.05). Journal of Investigative Dermatology 2010 130, 2638-2646DOI: (10.1038/jid.2010.191) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Plakophilin-1 (Pkp-1) affects cell survival after DNA damage. (a–c) Pkp-1 localization was observed in untreated A431 cells expressing Pkp-1 (a) or after 2hours incubation with 200μM etoposide (b) or 2mM hydroxyurea (c). Note the redistribution of Pkp-1 to nucleoli (bar in panel a=10μm). (d) UM-SCC-38 cells expressing short hairpin RNA targeting GFP (shGFP; top row) or Pkp-1 (shPkp-1; bottom row) were plated at low density and were treated with 20μM etoposide for 3hours, after which etoposide was removed. Foci were fixed, stained, and counted on day 12. (e). Statistical significance was determined using a two-tailed Student's t-test (P<0.05). Journal of Investigative Dermatology 2010 130, 2638-2646DOI: (10.1038/jid.2010.191) Copyright © 2010 The Society for Investigative Dermatology, Inc Terms and Conditions