Journal: International Journal of Molecular Sciences

Article Title: Expression of Alternative Splice Variants of 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase-4 in Normoxic and Hypoxic Melanoma Cells

doi: 10.3390/ijms22168848

Figure Lengend Snippet: The response of malignant melanoma cells to low oxygen concentration. ( A ) Upper panel: Malignant melanoma lines, WM115 and WM266-4, were cultured with 100 µM pimonidazole (hypoxyprobe) in normoxic (N) and hypoxic conditions (H) for 16 h. The formation of pimonidazole–protein adducts were detected using Western Blot. Lower panel: Ponceau S stained membrane is shown as an internal control for equal protein loading. ( B ) Upper panels: Melanoma cells were cultured for 16 h in normoxia and hypoxia. Then HIF-1 alpha subunit accumulation was verified using the Western Blot. β-actin is shown as an internal control for equal loading. Lower panels: Densitometry analysis of Western Blot bands intensity normalized to β-actin. Relative densitometry value is the average of four independent experiments. The mean ± SEM is shown. Student’s t -test was used to evaluate the influence of hypoxia on HIF-1 alpha subunit stabilization. * p < 0.05 by Student’s t -test, ** p < 0.01 by Student’s t -test, p -value between 0.05 and 0.1 by Student’s t -test was given as an indication of the trend. ( C ) CAIX and PFKFB4 expression was analyzed by RT-qPCR in both melanoma cell lines under hypoxic and normoxic conditions. Expression data for each transcript was normalized to that for the reference gene TBP. Means ± SEM of at least five independent experiments are presented relative to expression in normoxic controls. The Student t -test was used to evaluate the differences between normoxic and hypoxic expression of CAIX and PFKFB4 . ** p < 0.01 by Student’s t -test. ( D ) Upper panels: Melanoma cells were cultured for 16 h in normoxia and hypoxia. Then CAIX and PFKFB4 expression was verified using the Western Blot. β-actin is shown as an internal control for equal loading. Lower panels: Densitometry analysis of Western Blot bands intensity normalized to β-actin. Each relative densitometry value is the average of at least four independent experiments. The mean ± SEM is shown. Studen’st t -test was used to evaluate the influence of hypoxia on CAIX and PFKFB4 expression. * p < 0.05 by Student’s t -test, ** p < 0.01 by Student’s t -test, p -value between 0.05 and 0.1 by Student’s t -test was given as an indication of the trend.

Article Snippet: Two human melanoma cell lines WM115 and WM266-4 were obtained from Rockland Immunochemicals (Limerick, PA, USA).

Techniques: Concentration Assay, Cell Culture, Western Blot, Staining, Membrane, Control, Expressing, Quantitative RT-PCR

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: