Journal: Cells

Article Title: Tissue Inhibitor of Metalloproteinases-1 Overexpression Mediates Chemoresistance in Triple-Negative Breast Cancer Cells

doi: 10.3390/cells12131809

Figure Lengend Snippet: TIMP-1 expression is upregulated in chemoresistant TNBC cell lines. ( A ) Lysates from TNBC MDA-MB-231 and BT-549 cells and TPBC BT-474 cells were immunoblotted with anti-TIMP-1 antibody. Equal loading was confirmed by immunoblot with anti-vinculin antibody. ( B ) Cis-Pt-R and Dox-R cells exhibit increased fold changes in EC50 with respect to parental MDA-MB-231 cells. Following 48 h of Dox or Cis-Pt treatment, cell viability was determined and expressed as percentage of viable treated cells with respect to untreated controls. EC50 was estimated on the basis of three independent experiments. ** p < 0.01, *** p < 0.001; unpaired t -test. ( C ) Lysates from parental and chemoresistant cell lines were immunoblotted with anti-TIMP-1, anti-integrin αv, anti-PDGFRβ, anti-vimentin, anti-CD99, anti-CD44 and anti-ZO-1 antibodies, as indicated, by using anti-vinculin antibody as loading control; the histogram indicates the signal intensity of the bands, normalized to the respective anti-vinculin signal. Bars depict means ± SD of three independent experiments. ** p < 0.01, **** p < 0.0001 relative to MDA-MB-231; unpaired t -test. In ( A , C ), molecular weights of indicated proteins are reported.

Article Snippet: For assessing the effect of exogeneous TIMP-1 on cell viability, MDA-MB-231 (5000 cells/well, 96-well plates) and MDA-MB-231 cells interfered for CD63 expression (7000 cells/well, 96-well plates) were left untreated or treated for 48 h with 1 μM Dox or 20 µM Cis-Pt alone or in combination with either recombinant TIMP-1 (25 ng/mL) (Biotechne, Minneapolis, MN, USA) or TIMP-1 enriched conditioned media (CM) obtained from Dox-R cells.

Techniques: Expressing, Western Blot

Journal: Cells

Article Title: Tissue Inhibitor of Metalloproteinases-1 Overexpression Mediates Chemoresistance in Triple-Negative Breast Cancer Cells

doi: 10.3390/cells12131809

Figure Lengend Snippet: Exogenous TIMP-1 confers chemoresistance to MDA-MB-231 cells. ( A ) The conditioned medium obtained from MDA-MB-231, BT-549, Cis-Pt-R, Dox-R, BT-474 and MCF10A cells was analyzed by immunoblotting using anti-TIMP-1 antibody. FBS was used to exclude the presence of TIMP-1 in the serum supplementing the culture medium. ( B ) MDA-MB-231 cells were left untreated or treated for 48 h with 1 µM Dox or 20 µM Cis-Pt alone or in combination with either recombinant TIMP-1 (rTIMP-1) or TIMP-1-enriched CM obtained from Dox-R cells. Cis-Pt-R and Dox-R cells left untreated or treated with 20 µM Cis-Pt or 1 µM Dox, respectively, were used as chemoresistant reference cells. ( C ) MDA-MB-231 cells were treated for 48 h with rTIMP-1 plus 1 µM Dox or 20 µM Cis-Pt, in the presence or in the absence of 10 µM LY294002. In ( B , C ), cell viability was analyzed and expressed as percent of viable treated cells with respect to untreated cells. Bars depict means ± SD of three independent experiments. * p < 0.05; ** p < 0.01, relative to treated MDA-MB-231 cells; ns , not significant; °° p < 0.01, one-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: For assessing the effect of exogeneous TIMP-1 on cell viability, MDA-MB-231 (5000 cells/well, 96-well plates) and MDA-MB-231 cells interfered for CD63 expression (7000 cells/well, 96-well plates) were left untreated or treated for 48 h with 1 μM Dox or 20 µM Cis-Pt alone or in combination with either recombinant TIMP-1 (25 ng/mL) (Biotechne, Minneapolis, MN, USA) or TIMP-1 enriched conditioned media (CM) obtained from Dox-R cells.

Techniques: Western Blot, Recombinant, Comparison

Journal: Cells

Article Title: Tissue Inhibitor of Metalloproteinases-1 Overexpression Mediates Chemoresistance in Triple-Negative Breast Cancer Cells

doi: 10.3390/cells12131809

Figure Lengend Snippet: TIMP-1 binds to CD63 cell surface receptor. ( A ) Cell membrane and cytosolic fractions from the indicated cell lines were analyzed by immunoblotting using anti-TIMP-1 antibody. The cell-membrane receptor ITPRIPL1 and the cytosolic protein NF-kB-p65 were used as controls for the extraction procedure. ( B ) Confocal microscopic analysis of Cis-Pt-R cells co-stained with anti-TIMP-1 (green) and anti-CD63 (red) antibodies. Nuclei are visualized in blue. Magnification 63×, 1.0× digital zoom, scale bar = 10 μm. The merged image shows the overlay (yellow) of the two channels; white arrows indicate some points of colocalization between CD63 and TIMP-1. The representative section indicated by the dashed line was used for fluorescence intensity profiling analysis. Intensity peaks representing the red (CD63) and green (TIMP-1) emission wavelengths indicate areas of colocalization (black arrows). ( C ) MDA-MB-231 cells were transfected with si-CD63 or siRNA ctrl. Left: at 24 h post-transfection, cells were harvested, and cell lysates prepared and immunoblotted with anti-CD63 antibody. Anti-vinculin antibody was used as a loading control. Molecular weights of indicated proteins are reported. Middle: the histogram indicates the CD63/vinculin ratio of the densitometric signals. Values are shown relative to siRNA ctrl, arbitrarily set to 1. Bars depict means ± SD of three independent experiments. ** p < 0.01 relative to siRNA ctrl; unpaired t -test. Right: at 24 h post-transfection, cells were treated with 20 µM Cis-Pt alone or in combination with rTIMP-1. Cell viability was analyzed and expressed as percentage of viable treated cells compared to untreated cells. Bars depict means ± SD of three independent experiments. **** p < 0.0001, ### p < 0.001; ns , not significant; one-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: For assessing the effect of exogeneous TIMP-1 on cell viability, MDA-MB-231 (5000 cells/well, 96-well plates) and MDA-MB-231 cells interfered for CD63 expression (7000 cells/well, 96-well plates) were left untreated or treated for 48 h with 1 μM Dox or 20 µM Cis-Pt alone or in combination with either recombinant TIMP-1 (25 ng/mL) (Biotechne, Minneapolis, MN, USA) or TIMP-1 enriched conditioned media (CM) obtained from Dox-R cells.

Techniques: Cell Surface Receptor Assay, Membrane, Western Blot, Extraction, Staining, Fluorescence, Transfection, Comparison