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: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Cell titer blue proliferation assay of immortalized mesenchymal stem cells (MSCs), HEK-293, medulloblastoma (DAOY), and human basal cell carcinoma (BCC) cells stably expressing empty vector (control), wild-type FHL2 (FHL2), wild-type GLI2 (GLI2), truncated GLI2 (tGLI2), or the FHL2-GLI2 fusion. b Representative images of colony formation assay of MSCs, HEK-293, DAOY, and BCC cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 (scale bars, 5 mm; top). Quantification of the number of colonies/well compared to control (bottom). c Wound healing assay of MSCs, HEK-293, DAOY, and BCC cells stably expressing control, FHL2, GLI2, tGLI2 or FHL2-GLI2. The migratory effects/wound area was assessed at 0 and 24 h (Scale bar, 500 μm; top) and quantified (bottom). In a – c , data are representative of at least three independent experiments. Error bars, s.d. of mean; n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t -test.

Article Snippet: BCC cells were cultured in Human Basal Cell Carcinoma Cell Line Complete Media (CELPROGEN).

Techniques: Proliferation Assay, Stable Transfection, Expressing, Plasmid Preparation, Colony Assay, Wound Healing Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Differential gene expression analysis of human sclerosing stromal tumors (SSTs) subjected to RNA-sequencing ( n = 8, this study) and high-grade serous ovarian carcinomas ( n = 16; The Cancer Genome Atlas) and other sex cord-stromal tumors (SCSTs, n = 11; this study). Gene expression fold-change is color-coded according to the legend. Only genes significantly differentially expressed ( P < 0.05; two-tailed unpaired t -test) are shown. CPM, count per million. b Expression levels of Sonic Hedgehog (SHH) pathway genes in human SSTs ( n = 11) and other sex-cord stromal tumors ( n = 9) as defined using NanoString. Expression levels and SHH enrichment scores are color-coded according to the legends. *** P < 0.001, Wilcoxon rank test. Hierarchical clustering was performed using complete linkage and Euclidian distance. c Quantitative assessment of the Sonic Hedgehog pathway PTCH1 and GLI1 transcripts in immortalized mesenchymal stem cells (MSCs), HEK-293 and medulloblastoma (DAOY) cells and of PTCH1 and CCND1 transcripts in human basal cell carcinoma (BCC) cells stably expressing empty vector (control), wild-type FHL2 (FHL2), wild-type GLI2 (GLI2), truncated GLI2 (tGLI2), or FHL2-GLI2. Expression levels were normalized to GAPDH expression, and comparisons of mRNA expression levels were performed relative to control. d Representative western blot analysis of PTCH1 and GLI1 protein expression in MSC, HEK-293 and DAOY cells and of PTCH1 and CCND1 in BCC cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2. Tubulin was used as protein loading control. Quantification (below) of protein levels as compared to control. In c – d , data are representative of at least three independent experiments. Error bars, s.d. of mean; n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t -test.

Article Snippet: BCC cells were cultured in Human Basal Cell Carcinoma Cell Line Complete Media (CELPROGEN).

Techniques: Expressing, RNA Sequencing Assay, Two Tailed Test, Stable Transfection, Plasmid Preparation, Western Blot

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Representative confocal micrographs of immunofluorescence analysis of FLAG (red), 4–6-diamidino-2-phenylindole (DAPI, blue), and GFP (green) in HEK-293 cells stably expressing empty vector (control), wild-type FHL2 (FHL2), wild-type GLI2 (GLI2), truncated GLI2 (tGLI2), or FHL2-GLI2. Scale bars, 10 μm. b GLI response element (GLI-RE) luciferase reporter assay of HEK-293 cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 (top), and GLI-RE promoter activity in HEK-293 transiently transfected with control, FHL2-GLI2 and FHL2-GLI2 with R338A-K339A mutations in the Zinc Finger 5 of GLI2 required for DNA binding (FHL2-GLI2 Mutated; bottom). SV40-Renilla was used to normalize transfection efficiency. c Immunoprecipitation assay with SUFU antibody of HEK-293 cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2. Western blot analysis using anti-FLAG and anti-SUFU antibodies, and tubulin as loading control (left). GLI-RE promoter activity in HEK-293 stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 transfected with SUFU or control (right). d Cell titer blue proliferation assay of HEK-293, DAOY and BCC cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 treated with 20 µM GANT61 or vehicle control (DMSO). GANT, GANT61. e FLAG Chromatin Immunoprecipitation (ChIP) assay of GLI1 and PTCH1 promoters (promoter 1 and 2) in MSC and HEK-293 cells stably expressing either control or FHL2-GLI2. GLI1 and PTCH1 gene body and MYOD1, gene promoters not under GLI regulation, were used as negative controls. In b – d , data are representative of at least three independent experiments. Error bars, s.d. of mean; n.s., not significant; *P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t -test.

Article Snippet: BCC cells were cultured in Human Basal Cell Carcinoma Cell Line Complete Media (CELPROGEN).

Techniques: Immunofluorescence, Stable Transfection, Expressing, Plasmid Preparation, Luciferase, Reporter Assay, Activity Assay, Transfection, Binding Assay, Immunoprecipitation, Western Blot, Proliferation Assay, Chromatin Immunoprecipitation, Two Tailed Test

Journal: Nature Communications

Article Title: Identification of recurrent FHL2-GLI2 oncogenic fusion in sclerosing stromal tumors of the ovary

doi: 10.1038/s41467-019-13806-x

Figure Lengend Snippet: a Cell titer blue proliferation assay of immortalized mesenchymal stem cells (MSCs), HEK-293, medulloblastoma (DAOY) and human basal cell carcinoma (BCC) cells stably expressing empty vector (control), wild-type FHL2 (FHL2), wild-type GLI2 (GLI2), truncated GLI2 (tGLI2) or FHL2-GLI2 treated with 250 nM Vismodegib or vehicle control (DMSO). b Representative images of colony formation assay of MSC, HEK-293, DAOY, and BCC cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 treated with Vismodegib 500 nM or vehicle control (DMSO). Scale bars, 5 mm. Quantification of the number of colonies/well compared to control (bottom). c Wound healing assay of MSC, HEK-293, DAOY, and BCC cells stably expressing control, FHL2, GLI2, tGLI2, or FHL2-GLI2 treated with 250 nM Vismodegib or vehicle control (DMSO). The migratory effect/wound area was assessed at 0 and 24 h and quantified compared to DMSO (bottom). Vismo, Vismodegib. Scale bars, 500 μm. In a – c , data are representative of at least three independent experiments. Error bars, s.d. of mean; n.s., not significant; * P < 0.05, ** P < 0.01, *** P < 0.001; two-tailed unpaired t -test.

Article Snippet: BCC cells were cultured in Human Basal Cell Carcinoma Cell Line Complete Media (CELPROGEN).

Techniques: Proliferation Assay, Stable Transfection, Expressing, Plasmid Preparation, Colony Assay, Wound Healing Assay, Two Tailed Test