Journal: International Journal of Molecular Sciences

Article Title: Cell State Transitions and Phenotypic Heterogeneity in Luminal Breast Cancer Implicating MicroRNAs as Potential Regulators

doi: 10.3390/ijms24043497

Figure Lengend Snippet: Expression profiling of stemness-associated markers in breast cancer. ( A ) Breast cancer cell lines were screened for the expression of the cell surface marker CD147/Basigin or EMMPRIN, CD151/tetraspanin, and drug transporter proteins CD243/ABCB1 and CD338/ABCG2. More than 5% of MCF7, BT474, and SKBR3 cells expressed drug transporter proteins (* comparison between unstained control and stained cells for each cell line, p ≤ 0.05; calculated by Student’s t -test). ( B ) Immunofluorescence staining for CD147 and CD151 (scale bar measures 75 µm). ( C ) Breast cancer cell lines MCF7, BT474, and SKBR3 showed positive expression for aldehyde dehydrogenase activity (ALDH), indicating the enrichment of cells with higher self-renewal and drug resistance potential.

Article Snippet: The panel of antibodies used for phenotypic characterization were CD147-FITC (Clone REA282), CD151-PE (Clone REA265), CD243/ABCB1-Viobright FITC (Clone REA495), and CD338/ABCG2-PerCP_Vio700 (Clone 5D3), purchased from Miltenyi Biotec, Gaithersburg, MD, USA).

Techniques: Expressing, Marker, Comparison, Control, Staining, Immunofluorescence, Activity 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 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: Glia

Article Title: Endoplasmic Reticulum Stress Amplifies Cytokine Responses in Astrocytes via a PERK / eIF2α / JAK1 Signaling Axis

doi: 10.1002/glia.70067

Figure Lengend Snippet: JAK1 and PERK are required to augment TNF‐α induced gene expression in human glioma cells. (A) 1321N1 cells stably expressing Cas9 were transfected with non‐targeting (NT) or JAK1 guide RNAs (gRNA) to establish non‐clonal cell lines. These cells were then treated with IFN‐γ (10 ng/mL) for 30 min followed by immunoblotting. (B) NT and JAK1 gRNA (#2) cells were treated with thaps (1 μM), TNF‐α (10 ng/mL), or both for 4 h followed by qPCR. (C) 1321N1 cells stably expressing Cas9 were transfected with NT or PERK gRNA to establish non‐clonal cell lines. These cells were then treated with thaps (1 μM) for the indicated times followed by immunoblotting. (D) NT and PERK gRNA cells were treated with thaps (1 μM), TNF‐α (10 ng/mL), or both for 4 h followed by qPCR. N = 4, data are means ± standard deviation.

Article Snippet: 1321N1 human astrocytoma cell line stably expressing Cas9 nuclease was purchased from GeneCopoeia.

Techniques: Gene Expression, Stable Transfection, Expressing, Transfection, Western Blot, Standard Deviation