Journal: NPJ Breast Cancer

Article Title: NDRG4 promoter hypermethylation is a mechanistic biomarker associated with metastatic progression in breast cancer patients

doi: 10.1038/s41523-019-0106-x

Figure Lengend Snippet: NDRG4 depletion in breast tumor cells increases lymph node adhesion and cell migration toward VN. a NDRG4 expression meta-analysis in human breast cancer cell lines using GOBO application . Breast cancer subtypes can be identified by different colors: red = Basal A, gray = Basal B and blue = Luminal cells. b Analysis of NDRG4 mRNA expression levels in MCF-7 cells transfected with NDRG4-shRNAs or scramble control (shSCR). Expression levels are relative to wild type cells and normalized to hydroxymethylbilane synthase (HMBS) gene. Error bars represent SEM of biological replicates ( n = 5). *** p < 0.001, ns = not significant, by one-way ANOVA. c Western blot analysis of NDRG4 expression in cytoplasmic extracts of MCF-7 shNDRG4s or shSCRs cells, using antibodies against NDRG4 and Actin. MCF-7 cells express all the three isoforms of NDRG4: 37 kDa (NDRG4-B), 39 kDa (NDRG4-B var ) and 41 kDa isoform (NDRG4-H). For simplicity, results obtained for two independent shNDRG4 clones were grouped and presented as the shNDRG4 group. ( d , left) Representative images of adherent red fluorescent MCF-7 cells on frozen rat lymph node sections. d , e Quantification of adherent MCF7 ( d ) and T47D ( e ) breast tumor cells in lymph nodes sections (right, n = 4 independent experiments for MCF-7 and 3 for T47D, ** p < 0.01 by two-tailed t test). f Stationary adhesion assays showed that NDRG4 knockdown promotes ‘adhesive switch’ between FN and VN. Error bars represent SEM of biological replicates ( n = 4). Cells do not adhere to albumin control (BSA, data not shown). * p < 0.01, ** p < 0.01, ns = not significant by two-tailed t tests. g , h Haptotactic cell migration toward VN was analyzed by transwell migration assay in MCF-7 ( g ) and T47D ( h ) cell lines. Error bars represent SEM of biological replicates ( n = 4). *** p < 0.001 by two-tailed t tests

Article Snippet: For stable transfection of NDRG4 shRNA into MCF-7 and T47D breast tumor cells, the pGFP-V-RS vector containing shRNA inserts which targeted the 3′-unttranslated region of NDRG4 (TRCN0000134583 and TRCN0000137216) was purchased (Origene).

Techniques: Migration, Expressing, Transfection, Western Blot, Clone Assay, Two Tailed Test, Transwell Migration Assay

Journal: NPJ Breast Cancer

Article Title: NDRG4 promoter hypermethylation is a mechanistic biomarker associated with metastatic progression in breast cancer patients

doi: 10.1038/s41523-019-0106-x

Figure Lengend Snippet: NDRG4 knockdown promotes clustering of β1-integrin at the leading edge of T47D cells. Representative confocal images of β1 integrin subunit (MAB1965, green) at the ventral cell surface of VN-adherent T47D shNDRG4 or shSCR cells

Article Snippet: For stable transfection of NDRG4 shRNA into MCF-7 and T47D breast tumor cells, the pGFP-V-RS vector containing shRNA inserts which targeted the 3′-unttranslated region of NDRG4 (TRCN0000134583 and TRCN0000137216) was purchased (Origene).

Techniques:

Journal: Cancers

Article Title: Implication of COPB2 Expression on Cutaneous Squamous Cell Carcinoma Pathogenesis

doi: 10.3390/cancers14082038

Figure Lengend Snippet: Effect of COPB2 knockdown on biologic behavior of cutaneous squamous cell carcinoma (cSCC) cells: ( A ) (i) COPB2 protein expression decreased predominantly after COPB2 knockdown in both HSC-1 and A431 cells (Magnification, 400×; Scale bar, 50 μm); (ii) Proliferation ability of both HSC-1 and A431 cells decreased significantly after COPB2 knockdown at the indicated time points (Both p < 0.001, Mann-Whitney U-test). ( B ) (i) Representative patterns of cell invasion in each group of cSCC cells (Magnification, 100×; Scale bar, 200 μm). (ii) Invasion ability of both HSC-1 and A431 cells decreased significantly after COPB2 knockdown (Both p = 0.002, Mann-Whitney U-test). ( C ) Apoptotic cells increased predominantly after COPB2 knockdown in both HSC-1 and A431 cells. ( D ) (i) Tumor nodules of xenograft mouse models obtained from control and shCOPB2 groups of A431 cells (Scale bar, 1 cm). (ii) Volume of the tumor nodules increased significantly in control group compared to that in the COPB2 knockdown group at the indicated days (* p = 0.008, Mann-WhitneyU-test). (iii) Representative patterns of apoptotic cells in tumor nodules from each group of A431 cells-xenograft models (Magnification, 400×; Scale bar, 50 μm). Apoptotic cells were predominantly higher in the COPB2 knockdown group than in the control group in A431 cells ( p < 0.001, Mann-Whitney U-test).

Article Snippet: COPB2 knockdown stable HSC-1 and A431, as well as related control cells, were established using pGFP-C-shCOPB2 lentiviral particles (OriGene, Rockville, MD, USA) and maintained in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% fetal bovine serum (FBS; Invitrogen, Waltham, MA, USA) and 1% streptomycin/penicillin (Gibco, Waltham, MA, USA).

Techniques: Expressing, MANN-WHITNEY

Journal: Oncotarget

Article Title: The metastasis suppressor CD82/KAI1 inhibits fibronectin adhesion-induced epithelial-to-mesenchymal transition in prostate cancer cells by repressing the associated integrin signaling

doi: 10.18632/oncotarget.13767

Figure Lengend Snippet: A. Human prostate epithelial (PZ-HPV-7) and cancer (DU145, LNCaP, and PC3) cell lines and stably CD82-transfected DU145 and antisense CD82 fragment-transfected LNCaP cell clones were assessed for CD82 expression levels through immunoblotting analysis. B. Stably CD82-transfected DU145 and antisense CD82 fragment-transfected LNCaP cell clones grown on fibronectin (FN) were viewed under a phase-contrast microscope. Scale bar, 10 μm. C. The cells were seeded onto plates precoated with poly-L(+)-lysine (p-Lys) or FN and cultured for the indicated time periods. Expression of E-cadherin and mesenchymal marker proteins was examined through immunoblotting analysis using antibodies specific to each protein.

Article Snippet: PZ-HPV-7 (ATCC), a human prostate epithelial cell line transformed by human papillomavirus-18, was cultured in K-SFM medium (Invitrogen, Carlsbad, CA) supplemented with 0.05 mg/ml bovine pituitary extract and 5 ng/ml EGF.

Techniques: Stable Transfection, Transfection, Clone Assay, Expressing, Western Blot, Microscopy, Cell Culture, Marker

Journal: Oncotarget

Article Title: The metastasis suppressor CD82/KAI1 inhibits fibronectin adhesion-induced epithelial-to-mesenchymal transition in prostate cancer cells by repressing the associated integrin signaling

doi: 10.18632/oncotarget.13767

Figure Lengend Snippet: A. PZ-HPV-7 prostate epithelial cells were lysed with Brij 97 detergent, and immunoprecipitation (IP) was performed with normal mouse IgG or anti-CD82 antibody. The immunoprecipitates were analyzed by immnublotting using anti-integrin β 1 , α 3 , α 5 , or α 6 antibody. B. CD82 mutant cDNA, which encodes CD82 with a large extracellular loop (LEL) substituted with that of TM4SF2 as illustrated, was generated by PCR and subcloned into the pAdEasy-1 adenoviral vector to produce recombinant adenovirus. C. CD82-deficient PC3 prostate cancer cells grown on fibronectin (FN) were infected with adenovirus containing a wild-type (wt) or mutant (mt) CD82 expression construct, and Brij 97 detergent lysates were subjected to immunoprecipitation with an anti-β 1 integrin antibody followed by immunoblotting analysis using antibodies that recognize the C-terminus or LEL of CD82 and the LEL of TM4SF2. D. PC3 cells grown on poly-L(+)-lysine (p-Lys) or FN were infected with adenovirus containing a wt- or mt-CD82 expression construct and then assessed for the protein levels of E-cadherin and Snail. E. PC3 cells grown on FN were infected with wt-CD82 construct-containing adenovirus either alone or together with mt-CD82 construct-containing adenovirus and examined for E-cadherin and Snail expression. Numbers in parentheses represent the MOI values of adenovirus.

Article Snippet: PZ-HPV-7 (ATCC), a human prostate epithelial cell line transformed by human papillomavirus-18, was cultured in K-SFM medium (Invitrogen, Carlsbad, CA) supplemented with 0.05 mg/ml bovine pituitary extract and 5 ng/ml EGF.

Techniques: Immunoprecipitation, Mutagenesis, Generated, Plasmid Preparation, Recombinant, Infection, Expressing, Construct, Western Blot

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: Long Non-coding RNA HOXA11 Antisense Promotes Cell Proliferation and Invasion and Predicts Patient Prognosis in Serous Ovarian Cancer

doi: 10.4143/crt.2016.263

Figure Lengend Snippet: Knockdown of HOXA11 antisense ( HOXA11as ) inhibits serous ovarian cancer cell proliferation. (A) Expression of HOXA11as in human ovarian surface epithelial cell line (HOSE) and six ovarian cancer cell lines determined by quantitative real time polymerase chain reaction (qRT-PCR). (B) Knockdown efficiency was determined by qRT-PCR analysis in OVCA429 and SKOV3 cells. Cells were transfected with HOXA11as -specific siRNA (siHOXA11as) and negative control siRNA (siNC). (C, D) Knockdown of HOXA11as significantly reduced cell proliferation in OVCA429 and SKOV3 cells as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Bars indicate mean±standard deviation of three independent experiments performed in triplicate. * p < 0.05 vs. siNC. siHOXA11as, HOXA11as -specific siRNA.

Article Snippet: Normal human ovarian surface epithelial (HOSE) cells were purchased from ScienCell Research Laboratories (San Diego, CA).

Techniques: Knockdown, Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Transfection, Negative Control, MTT Assay, Standard Deviation