Journal: PLoS ONE

Article Title: The Desmosomal Armadillo Protein Plakoglobin Regulates Prostate Cancer Cell Adhesion and Motility through Vitronectin-Dependent Src Signaling

doi: 10.1371/journal.pone.0042132

Figure Lengend Snippet: A. Prostate cancer cell lines were lysed and subjected to SDS-PAGE, followed by immunoblotting with antibodies against PG and GAPDH. PG levels were determined by normalizing to GAPDH levels for each cell line using Image J. The immunoblot shows that the expression of PG in prostate cancer cell lines inversely correlates with the degree of aggressiveness of the cell line. B. Prostate cancer cell lines were plated onto coverslips and allowed to attach and spread. Immunofluorescence was performed using antibodies against PG to demonstrate the localization of PG in ARCaP E , ARCaP M , LNCaP, C4-2B, DU145, PC-3, and PC3-M cell lines. The immunofluorescence shows weaker staining and a loss of cell-cell border localization of PG in the more aggressive cell lines (ARCaP M , C4-2B, and PC3-M). Representatives of at least three independent immunoblots are shown, with numbers representing GAPDH normalized PG protein levels for the blot shown. All the PG protein levels were normalized to ARCaP E levels within each blot. The average level and standard deviation of PG protein levels in panel A is as follows: ARCaP E 1.0, ARCaP M 0.3+/−0.2, LNCaP 1.5+/−0.5, C4-2B 0.5+/−0.2, DU145 3.4+/−1.2, PC-3 1.0+/−0.4, PC3-M 0.5+/−0.2.

Article Snippet: PC3-M cells, LNCaP cells, PC-3 cells (all gifts from Dr. Raymond C. Bergan, Northwestern University, Chicago, IL), DU145 cells (a gift from Dr. Lester F. Lau, University of Illinois, Chicago, IL) and C4-2B cells (a gift from Dr. R.S.

Techniques: SDS Page, Western Blot, Expressing, Immunofluorescence, Staining, Standard Deviation

Journal: PLoS ONE

Article Title: The Desmosomal Armadillo Protein Plakoglobin Regulates Prostate Cancer Cell Adhesion and Motility through Vitronectin-Dependent Src Signaling

doi: 10.1371/journal.pone.0042132

Figure Lengend Snippet: A. Representative image of a dispase assay in PC3-M cells after transduction with PG OE adenovirus or control GFP adenovirus. B. Quantitations of dispase assays performed in triplicate; LNCaP, C4-2B, or PC3-M cells were plated in 6-well plates, and allowed to attach and spread. Cultures were transduced with GFP-containing adenovirus (GFP) or PG-containing adenovirus (PG OE), and after 24 hours the dispase assay was performed. Overexpression of PG in all 3 cell lines strengthens cell-cell adhesion. C. LNCaP cells were plated in 6-well plates and allowed to attach and spread. The cells were then transfected with control siRNA or PG siRNA pool. The dispase assay was performed 96 hours after transfection. Suppression of PG expression results in a weakening of cell-cell adhesion in LNCaP cells. D. Representative image of a hanging drop assay in DU145 cells after transfection with control or PG siRNA pool. E–F. Quantitation of hanging drop assays performed in triplicate; C4-2B and PC3-M cells were transduced with GFP- or PG-containing adenoviruses (E), ARCaP E, LNCaP, DU145 and PC3 cells were transfected with control siRNA or PG siRNA pool (F). Aggregation assays were done 24 h (E), or 96 h (F) after the treatment. Graphs represent averages +/− SEM. *P<0.06; **P<0.005; ***P<0.0007, by paired Student t test.

Article Snippet: PC3-M cells, LNCaP cells, PC-3 cells (all gifts from Dr. Raymond C. Bergan, Northwestern University, Chicago, IL), DU145 cells (a gift from Dr. Lester F. Lau, University of Illinois, Chicago, IL) and C4-2B cells (a gift from Dr. R.S.

Techniques: Transduction, Control, Over Expression, Transfection, Expressing, Quantitation Assay

Journal: PLoS ONE

Article Title: The Desmosomal Armadillo Protein Plakoglobin Regulates Prostate Cancer Cell Adhesion and Motility through Vitronectin-Dependent Src Signaling

doi: 10.1371/journal.pone.0042132

Figure Lengend Snippet: A. PC3-M and ARCaP M were plated in 24-well plates and allowed to attach and spread. The cells were then transduced with GFP-containing adenovirus or PG-containing adenovirus, and after 24 hours a scratch wound was made. Images were taken at 0 and 24 hours and the % wound closure was calculated by comparing the size of the wound at 24 hours to the 0 hour time point for each condition. Overexpression of PG suppresses motility in these two cell lines. B. ARCaP E and C4-2B cells were plated in 24-well plates and allowed to attach and spread. The cells were then transfected with control siRNA or PG siRNA pool, and 72 hours after transfection, the scratch wounds were made. Suppression of PG leads to an increase in motility in ARCaP E and C4-2B cells. Scratch wound assay done in triplicate in prostate cancer cell lines after overexpression or knockdown of PG. C. ARCaP M , C4-2B, DU145 and PC3-M cells were transduced with GFP-containing adenovirus or PG-containing adenovirus, and after 24 hours plated onto Matrigel coated transwell membranes for an invasion assay. Overexpression of PG suppresses invasion in these four cell lines. D. ARCaPE, LNCaP, C4-2B, DU145, PC-3 and PC3-M cells were transfected with control siRNA or PG siRNA pool, and 72 hours after transfection, plated onto Matrigel coated transwell membranes for an invasion assay. Suppression of PG leads to an increase in invasion in these six cell lines. Invasion assays were done in triplicate. Average of the total number of invading cells is presented. Graphs represent averages +/− SEM. *P<0.1; **P<0.05; ***P<0.001, by paired Student t test.

Article Snippet: PC3-M cells, LNCaP cells, PC-3 cells (all gifts from Dr. Raymond C. Bergan, Northwestern University, Chicago, IL), DU145 cells (a gift from Dr. Lester F. Lau, University of Illinois, Chicago, IL) and C4-2B cells (a gift from Dr. R.S.

Techniques: Transduction, Over Expression, Transfection, Control, Scratch Wound Assay Assay, Knockdown, Invasion Assay

Journal: PLoS ONE

Article Title: The Desmosomal Armadillo Protein Plakoglobin Regulates Prostate Cancer Cell Adhesion and Motility through Vitronectin-Dependent Src Signaling

doi: 10.1371/journal.pone.0042132

Figure Lengend Snippet: A–B. PC3-M, C4-2B (A), and LNCaP cells (B) were plated in 6 well plates and allowed to attach and spread. LNCaP cells were transfected with control siRNA or PG siRNA (a pool of 4 sequences). PC3-M, C4-2B, and LNCaP cells were treated with medium containing PP2 (10 µM) or DMSO (solvent control) for 24 hours prior to performing the dispase assay. Inhibition of Src strengthens cell-cell adhesion in PCa cells in general and is able to rescue cell-cell adhesion in PG-deficient cells. C. PC3-M and ARCaP M cells were plated in 24-well plates and allowed to attach and spread. The cells were treated with medium containing the selective Src- family kinase inhibitor PP2 (10 µM) or DMSO (solvent control) for 24 hours prior to performing the scratch wound assay. D. ARCaP E cells were plated in 24-well plates and allowed to attach and spread. The cells were transduced with GFP-containing adenovirus or caSrc-containing adenovirus, and after 24 hours a scratch wound was made. Activity of Src is directly correlated with motility of PCa cells. E. ARCaP M , PC3-M, and LNCaP cells were plated in 6-well plates and allowed to attach and spread. LNCaP cells were transfected with control siRNA or PG siRNA pool and after 96 hours, the cells were lysed. ARCAP M and PC3-M cells were transduced with GFP-containing adenovirus or PG-containing adenovirus, and after 24 hours the cells were lysed. The lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies against PG, pSrc, Src, and GAPDH. Levels of pSrc were determined by normalizing to Src levels for each cell line using Image J. Phosphorylation (activation) of Src is inversely correlated with PG levels. Representatives of at least three independent immunoblots are shown, with numbers representing GAPDH normalized pSrc/Src ratio for the blot shown. The average ratio and standard deviation of pSrc/Src normalized for loading by GAPDH is as follows: ARCaP M 0.6+/−0.2, LNCaP 1.7+/−0.2, PC3M 0.7+/−0.1. Graphs represent averages +/− SEM. *P<0.04; **P<003; ****P<0.0001, by paired Student t test.

Article Snippet: PC3-M cells, LNCaP cells, PC-3 cells (all gifts from Dr. Raymond C. Bergan, Northwestern University, Chicago, IL), DU145 cells (a gift from Dr. Lester F. Lau, University of Illinois, Chicago, IL) and C4-2B cells (a gift from Dr. R.S.

Techniques: Transfection, Control, Solvent, Inhibition, Scratch Wound Assay Assay, Transduction, Activity Assay, SDS Page, Western Blot, Phospho-proteomics, Activation Assay, Standard Deviation

Journal: PLoS ONE

Article Title: The Desmosomal Armadillo Protein Plakoglobin Regulates Prostate Cancer Cell Adhesion and Motility through Vitronectin-Dependent Src Signaling

doi: 10.1371/journal.pone.0042132

Figure Lengend Snippet: A–E. ARCaP E , ARCaP M , LNCaP, C4-2B, and PC3-M cells were plated in 6-well plates and allowed to attach and spread. The cells were either transduced with GFP- or PG-containing adenovirus and were lysed after 24 hours (PC3-M, C4-2B, ARCaP M ); or the cells were transfected with control siRNA or PG siRNA pool and after 96 hours lysed (ARCaP E , LNCaP, C4-2B). The lysates were subjected to SDS-PAGE followed by immunoblotting with antibodies against VN (A), FN (B), LN (C), Col. I (D), Col IV (E) and GAPDH. Levels of ECM proteins were determined by normalizing to GAPDH levels for each cell line using Image J. Representatives of at least three independent immunoblots are shown, with numbers representing GAPDH normalized PG siRNA/Con siRNA or PG OE/GFP ratio for the blot shown. The average ratio and standard deviation of GAPDH normalized PG siRNA/Con siRNA and PG OE/GFP is as follows: (A) ARCaP E 3.1+/−1.3, LNCaP 2.0+/−0.9, PC3-M 0.3+/−0.1; (B) LNCaP 0.7+/−0.05, C4-2B KD 0.8+/−0.1, C4-2B OE 1.2+/−0.2, PC3-M 1.5+/−0.05; (C) ARCaP M 0.6+/−0.1, PC3-M 0.4+/−0.3; (D) 0.3+/−0.1; (E) 0.4+/−0.2.

Article Snippet: PC3-M cells, LNCaP cells, PC-3 cells (all gifts from Dr. Raymond C. Bergan, Northwestern University, Chicago, IL), DU145 cells (a gift from Dr. Lester F. Lau, University of Illinois, Chicago, IL) and C4-2B cells (a gift from Dr. R.S.

Techniques: Transduction, Transfection, Control, SDS Page, Western Blot, Standard Deviation