Journal: BMC Cancer

Article Title: A novel FOXO1-mediated dedifferentiation blocking role for DKK3 in adrenocortical carcinogenesis

doi: 10.1186/s12885-017-3152-5

Figure Lengend Snippet: Reduced DKK3 expression in ACC. a Reduced DKK3 gene expression in 7 tumor samples (T1–T7) compared to 3 histologically normal adrenal samples (N1–N3). T5a and T5b: RNA from two different areas of one tumor. Magnitude of gene expression relative to housekeeping gene panel shown below. b Immunofluorescence detection of DKK3 and β-catenin in normal adrenal cortex ( a-g ) and ACC ( h-n ). Tissue sections treated with primary/secondary antibodies for DKK3 (FITC, green ; c, j ) or β-catenin (TR, red ; e , l ), DAPI ( blue for nuclear staining; b , i ), or combinations of FITC/DAPI ( d , k ), TR/DAPI ( f , m ), or FITC/TR/DAPI ( a , h , g , n ). a and h : 100× magnification; b - g , i - n : 400× magnification; inset ( g , n ): 1000× magnification. c DKK3 gene expression (fold-change) in 37 ACC samples relative to average expression of 14 normal adrenal samples normalized to 1. d Average DKK3 expression (fold-change) in study cohort ( n = 37) compared to average expression from 14 normal adrenal samples

Article Snippet: Myc-DDK tagged pCMV6-Entry, pCMV6-Entry/GFP, and pCMV6-Entry/ DKK3 plasmid vectors (Origene) were used for transient and stable expression.

Techniques: Expressing, Immunofluorescence, Staining

Journal: BMC Cancer

Article Title: A novel FOXO1-mediated dedifferentiation blocking role for DKK3 in adrenocortical carcinogenesis

doi: 10.1186/s12885-017-3152-5

Figure Lengend Snippet: DKK3 mRNA expression, promoter methylation, and gene copy number alterations in adrenocortical carcinoma

Article Snippet: Myc-DDK tagged pCMV6-Entry, pCMV6-Entry/GFP, and pCMV6-Entry/ DKK3 plasmid vectors (Origene) were used for transient and stable expression.

Techniques: Expressing, Methylation

Journal: BMC Cancer

Article Title: A novel FOXO1-mediated dedifferentiation blocking role for DKK3 in adrenocortical carcinogenesis

doi: 10.1186/s12885-017-3152-5

Figure Lengend Snippet: RNAi silencing of DKK3 in ACC cell lines and effects on cell behavior. a Western immunoblot detection of endogenous DKK3 in SW-13 ( left ) and NCI-H295R ( right ). b and c , Relative expression of DKK3 as determined by qRT-PCR in siRNA-treated SW-13 (2B) and NCI-H295R (2C) cells, normalized to expression in cells treated with scrambled siRNA for 24 h. d Western immunoblot detection of DKK3 in SW-13 cells treated with control (1), scrambled negative siRNA (2), 10 (3), 20 (4), and 40 nM (5) DKK3 siRNAs for 24 h followed by protein extraction 48 h post-transfection. e-h , NCI-H295R ( e and f ) or SW-13 ( g and h ) cells treated with Lipofectamine (Lipo), scrambled negative siRNA (S-ive), or DKK3 siRNA (DKK3) for 24 h, allowed to grow in clonogenic growth conditions ( e and g ), or allowed to migrate through modified Boyden chambers through growth factor concentration gradient for 12 ( f ) or 4 ( h ) hours. Clones with 12 ± 2 cells were fixed, stained, and counted with light microscope ( e and g ); cells that migrated to lower side of modified Boyden chamber membranes were fixed, stained, and counted ( f and h )

Article Snippet: Myc-DDK tagged pCMV6-Entry, pCMV6-Entry/GFP, and pCMV6-Entry/ DKK3 plasmid vectors (Origene) were used for transient and stable expression.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Protein Extraction, Transfection, Modification, Concentration Assay, Clone Assay, Staining, Light Microscopy

Journal: BMC Cancer

Article Title: A novel FOXO1-mediated dedifferentiation blocking role for DKK3 in adrenocortical carcinogenesis

doi: 10.1186/s12885-017-3152-5

Figure Lengend Snippet: ACC cells were either treated with exogenous recombinant DKK3 ( a ) or enforced to express Myc-DDK tagged DKK3 ( b ) and assayed for cell behaviors. a SW-13 N/D ( left ) or NCI-H295R ( right ) cells were untreated (SW N/D-, 295-) or treated (SW N/D+, 295+) with exogenous DKK3 for 24 h and allowed to migrate through modified Boyden chamber for 4 h. Cells migrating to lower surface were fixed, stained, and counted. b Western immunoblot detection of endogenous DKK3 and ectopically expressed DKK3 (Myc-DDK/DKK3) in vector control ( lane 1 ), SW-DKK3 ( lane 2 ), or Myc-DDK/GFP control ( lane 3 ) cells. c SW-13, SW-Neo, and SW-DKK3 cells plated in 24-well plates (5000 cells/well) were grown 8 days. Quadruplicate wells from each cell type were trypsinized, incubated in 0.2% Trypan blue , and viable cells were counted using hemocytometer. Data shown represent one of three independent experiments. d and e , Five thousand SW-13 or SW-DKK3 cells plated in 6-well plates were allowed to grow 7 days; clones were fixed, stained, and enumerated into 2 classes of ( a ) 12 ± 2 cells ( filled light grey ) and (b) 4 ± 2 cells ( filled black ). Majority of clones formed from SW-Neo cells were large ( e ; left ), while SW-DKK3 cells produced a significant number of small colonies (4 ± 2) comprised of large cells ( e ; right ). f One hundred thousand SW-13, SW-Neo, and SW-DKK3 cells were allowed to migrate through modified Boyden chamber for 4 h; cells that migrated to the lower side of the membrane were fixed, stained, and counted. g One hundred thousand SW-13, SW-Neo, and SW-DKK3 cells were allowed to invade through Matrigel in modified Boyden chambers for 24 h. Cells that invaded through Matrigel and migrated to the lower side of the membrane were fixed, stained, and counted

Article Snippet: Myc-DDK tagged pCMV6-Entry, pCMV6-Entry/GFP, and pCMV6-Entry/ DKK3 plasmid vectors (Origene) were used for transient and stable expression.

Techniques: Recombinant, Modification, Staining, Western Blot, Plasmid Preparation, Incubation, Clone Assay, Produced

Journal: BMC Cancer

Article Title: A novel FOXO1-mediated dedifferentiation blocking role for DKK3 in adrenocortical carcinogenesis

doi: 10.1186/s12885-017-3152-5

Figure Lengend Snippet: Constitutive over-expression of DKK3 reorganizes cellular extensions and cell spreading. a - c SW-13 ( a ), SW-Neo ( b ), and SW-DKK3 ( c ) cells were grown on glass cover-slips, fixed, stained, and photographed. SW-13 and SW-Neo cells show a predominance of filopodia ( red arrowheads ) around edges; SW-DKK3 shows more lobopodia ( small green arcs ), absence of lamellipodia ( blue arc ), and few filopodia around edges. While cells in a and b appear to be polarized with filopodia at leading edge and lamellipodia at lagging edge, SW-DKK3 cells ( c ) show evenly spread flat lobopodia with extensive spreading and absence of polarity. Photomicrographs are taken using light microscope at 400× magnification. d Average number of lamellipodia, filopodia, and lobopodia per cell calculated from manual counting of cell extensions. Twenty randomly taken (400× magnification) photomicrographs of SW-13, SW-Neo, and SW-DKK cells used for quantification. e One hundred thousand SW-13, SW-Neo, and SW-DKK3 cells/well of 6-well plates were allowed to grow overnight, detached at specified times, cells remaining attached were fixed, stained, and counted manually

Article Snippet: Myc-DDK tagged pCMV6-Entry, pCMV6-Entry/GFP, and pCMV6-Entry/ DKK3 plasmid vectors (Origene) were used for transient and stable expression.

Techniques: Over Expression, Staining, Light Microscopy

Journal: BMC Cancer

Article Title: A novel FOXO1-mediated dedifferentiation blocking role for DKK3 in adrenocortical carcinogenesis

doi: 10.1186/s12885-017-3152-5

Figure Lengend Snippet: FOXO1 silencing releases DKK3-mediated block of cell migration. Cells were treated with scrambled negative siRNA (Neg.) or FOXO1 siRNA (Si-RNA) for 24 h, trypsinized, and allowed to migrate for 4 h through modified Boyden chamber. Migrated cells were fixed, stained, and counted manually. Total number of SW-Neo cells treated with scrambled siRNA normalized to 100%, and relative change in migration of SW-Neo and SW-DKK3 cells treated with FOXO1 siRNA is shown in overlapping line graph on left Y-axis. Relative FOXO1 expression in SW-Neo and SW-DKK3 cells treated with FOXO1 siRNA, normalized to FOXO1 expression in SW-Neo cells treated with scrambled siRNA set at 100 shown as bars on right Y-axis

Article Snippet: Myc-DDK tagged pCMV6-Entry, pCMV6-Entry/GFP, and pCMV6-Entry/ DKK3 plasmid vectors (Origene) were used for transient and stable expression.

Techniques: Blocking Assay, Migration, Modification, Staining, Expressing