Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: (A) Cell homogenates ( H ) from the indicated cell lines were used to prepare cytosolic ( C ) and membrane ( M ) fractions. Equivalent amounts of each fraction (10 μg of proteins) were subjected to SDS-PAGE and immunoblotting using antibodies to the proteins indicated on the right. The position of molecular mass markers is indicated on the left. (B) Densitometric quantification of the immunoblot signal of the levels of GOLPH3 in the cell homogenates as shown in (A). (C) Densitometric quantification of the immunoblot signal of the levels of GOLPH3 in cytosolic ( C ) and membrane ( M ) fractions as shown in (A). Bar represents the mean ± standard deviation of the amount of immunoblot signal normalized with the signal for β-actin, and also for the total amount of protein in each fraction (for more details see ). *** P < 0.001; ns , not statistically significant.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Membrane, SDS Page, Western Blot, Standard Deviation

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: (A-C) Samples of a membrane fraction (70 μg of proteins) from MCF 10A (A), MCF7 (B), and MDA-MB-231 (C) cells were incubated on ice for 1 hour with either 10 mM Tris HCl pH 7.4 ( Control ), 1 M KCl in 10 mM Tris HCl pH 7.4 ( KCl ) or 0.2 M Na 2 CO 3 pH 11.3 ( Na 2 CO 3 ). After centrifugation, pelleted membranes ( P ) and extracted proteins in the supernatant ( S ) were processed by SDS-PAGE and immunoblotting using antibodies to the proteins indicated on the right. Syn16 , Syntaxin 16. The position of molecular mass markers is indicated on the left. (D-F) Densitometric quantification of the immunoblot signal of the levels of GOLPH3 in pellets ( P ) and supernatants ( S ) as shown in A-C of membranes incubated in control conditions (D), in 1 M KCl (E), or in 0.2 M Na 2 CO 3 (F). Bar represents the mean ± standard deviation of the amount of immunoblot signal. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Membrane, Incubation, Control, Centrifugation, SDS Page, Western Blot, Standard Deviation

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: NRK (A and B), MCF 10A (C and D), MDA-MB-231 (E and F), and MCF7 (G and H) cells were left untreated ( Control ) or treated with 5 μg/ml BFA for 60 min ( BFA ). Cells were fixed, permeabilized, and immunolabeled with rabbit polyclonal antibody to GOLPH3, mouse monoclonal antibody to GM130, and either sheep antibody to TGN38 (A and B) or sheep antibody to TGN46 (C to H). Secondary antibodies were Alexa-594-conjugated donkey anti-rabbit IgG (red channel), Alexa-488-conjugated donkey anti-mouse IgG (green channel), and Alexa-647-conjugated donkey anti-sheep IgG (blue channel). Stained cells were examined by fluorescence microscopy. Merging red, green, and blue channels generated the fourth image on each row; yellow indicates overlapping localization of the red and green channels, cyan indicates overlapping localization of the green and blue channels, magenta indicates overlapping localization of the red and blue channels, and white indicates overlapping localization of all three channels. Insets show 1.7x magnifications. Bar, 10 μm.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Control, Immunolabeling, Staining, Fluorescence, Microscopy, Generated

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: (A-C) MCF 10A (A), MDA-MB-231 (B), and MCF7 (C) cells transiently expressing GFP-GOLPH3 were held in a microscope stage at 37°C and examined by fluorescence microscopy. The time after initiation of imaging is shown in the bottom right corner of each panel in minutes:seconds. Images are representative of 15–20 videos of up to 200 seconds of recording. In B, filled arrows indicate a vesicular structure moving from the Golgi to the periphery of the cell. In C, filled arrows indicate a vesicular structure moving from the periphery of the cell to the Golgi area, and filled arrowheads indicate a tubular structure elongating from the Golgi. Empty arrows and empty arrowheads indicate the initial position of mobile structures. Bars, 5 μm. (D-F) The number of tubule-vesicular structures moving centrifugally (D), the number of tubular structures elongating from the Golgi (E), or the number of tubule-vesicular structures moving centripetally (F), were quantified from videos corresponding to 180 seconds of imaging. Bar represents the mean + standard deviation of the observed profiles (n = 15). * P < 0.05; *** P < 0.001.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Expressing, Microscopy, Fluorescence, Imaging, Standard Deviation

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: (A-C) MCF 10A (A), MDA-MB-231 (B), and MCF7 (C) cells transiently expressing GFP-GOLPH3 were held in a microscope stage at 37°C. The area indicated by a white dotted-line rectangle in each set of images was bleached with a 488-nm laser set to 100% power. The fluorescence recovery after photobleaching (FRAP) was tracked by laser confocal microscopy with the 488-nm laser set to 2% power. Images were acquired before bleaching ( Pre-bleaching ), immediately after bleaching ( Bleaching ), and during the recovery of the fluorescence ( Recovery ) at approximately every 0.4-sec. Images of a representative experiment performed on each cell line are shown in each set of panels. Two images of the recovery of fluorescence are depicted with the time indicated in parenthesis in seconds. Bar, 10 μm. (D) Plot of the FRAP analysis of GFP-GOLPH3 in MCF 10A (black circles; n = 10), MDA-MB-231 (white circles; n = 10), and MCF7 (white squares; n = 10) cells. P , pre-bleaching; B ; bleaching. For simplicity, error bars are not depicted. *** P < 0.001; ns , not statistically significant. The halftime ( t 1/2 ) of maximal fluorescence recovery is indicated on the right in seconds (s).

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Expressing, Microscopy, Fluorescence, Confocal Microscopy

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: Samples (30 μg of proteins) of rat liver cytosol ( Cyt ), rat liver Golgi membranes, and of cytosolic ( Cyt ) and membrane ( Memb ) fractions from the cell lines indicated at the right were analyzed by two-dimensional gel electrophoresis (2-D GE) and immunoblotting using antibody to GOLPH3. Samples of rat liver Golgi membranes, and of the cytosolic and membrane fractions of each cell line, were dephosphorylated with calf intestine alkaline phosphatase ( CIAP ) before processing for 2-D GE. The position of molecular mass markers is indicated on the left. The position of isoelectric point ( pI ) markers is indicated at the bottom. Red asterisks indicate the position of additional, less abundant, but distinct spots in the samples of MCF7 cells that have slightly slower electrophoretic mobility. Numbers indicate different acidic forms identified in immunoblot films subjected to different exposure times.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Membrane, Two-Dimensional Gel Electrophoresis, Electrophoresis, Western Blot

Journal: PLoS ONE

Article Title: Distinct Biochemical Pools of Golgi Phosphoprotein 3 in the Human Breast Cancer Cell Lines MCF7 and MDA-MB-231

doi: 10.1371/journal.pone.0154719

Figure Lengend Snippet: (A) Membranes with the spotted phospholipids indicated on the left were incubated with untreated, recombinant GOLPH3 ( GOLPH3 ) or with recombinant GOLPH3 in the presence of cytosolic proteins from the cell lines indicated on the top. Bound recombinant GOLPH3 was detected by immunoblotting with antibody to GOLPH3. LysoPtdA , lysophosphatidic acid; LysoPtdCho , lysophosphatidylcholine; PtdIns , phosphatidylinositol; PtdIns(3)P , phosphatidylinositol 3-phosphate; PtdIns(4)P , phosphatidylinositol 4-phosphate; PtdIns(5)P , phosphatidylinositol 5-phosphate; PtdEth , phosphatidylethanolamine; PtdCho , phosphatidylcholine; S1P , sphingosine 1-phosphate; PtdIns(3 , 4)P 2 , phosphatidylinositol 3,4-bisphosphate; PtdIns(3 , 5)P 2 , phosphatidylinositol 3,5-bisphosphate; PtdIns(4 , 5)P 2 , phosphatidylinositol 4,5-bisphosphate; PtdIns(3 , 4 , 5)P 3 , phosphatidylinositol 3,4,5-trisphosphate; PtdA , phosphatidic acid; PtdSer , phosphatidylserine; Blank , no lipid. (B) Densitometric quantification of the immunoblot signal of the levels of untreated, recombinant GOLPH3 bound to different phospholipids as shown in (A). (C) Densitometric quantification of the immunoblot signal of the levels of recombinant GOLPH3 bound to phosphatidylinositol 4-phosphate after incubation with cytosolic proteins of the indicated cell lines as shown in (A). * P < 0.05; *** P < 0.001.

Article Snippet: For the generation of GOLPH3 constructs, a cDNA encoding full-length human GOLPH3 (GenBank/EMBL/DDBJ accession number NM_022130) was acquired from OriGene Technologies (Rockville, MD), and used as a template.

Techniques: Incubation, Recombinant, Western Blot

Journal: Cell Death and Differentiation

Article Title: Synergistic apoptotic effect of miR-183-5p and Polo-Like kinase 1 inhibitor NMS-P937 in breast cancer cells

doi: 10.1038/s41418-021-00864-2

Figure Lengend Snippet: A Left: Renilla luciferase activity analyses in HEK 293 T cells co-transfected with psiCHECK2-PLK1-CDS, psiCHECK2-PLK1-3′UTR, or psiCHECK-PLK1-3′UTR-MUT + miR-183-5p or negative control miRNA (NC) (Ambion). Results are 48 h post transfection. Right: Renilla luciferase activity analyses in HEK 293 T cells co-transfected with psiCHECK2-PLK1-CDS or psiCHECK2-PLK1-3′UTR + miR-18a-3p, miR-100-5p, or negative control miRNA (NC). Data ( n = 3/group) are presented as mean + % SD. B Left: Immunoblots showing PLK1 expression following no treatment (NT), transfection with negative control (NC) or miR-183-5p. Right: RT-qPCR demonstrating expression levels of miR-183-5p following transfection (results are x10 3 ). C Left: Immunoblots showing PLK1 expression following no treatment (NT) or miR-183-5p, following transfection with PLK1 CDS with 3′UTR (CDS + 3′UTR) or PLK1 CDS only (CDS only). Right: RT-qPCR demonstrating expression levels of miR-183-5p following transfection (results are x10 3 for MDA-MB-231, BT549 and ZR-75-1, x10 for T47D). D Western blots show results of the rescue experiments (see material and methods) performed on the MDA-MB-231 (left) and T47D (right) cell lines. Bar graphs are densitometry results of PLK1-fold expression. Vinculin was used as internal control. Data are the mean of at least 3 independent experiments ( N = 3) + SD. ** p -value <0.01 compared to negative control by Student’s unpaired t test. E TCGA, IBCD analysis. Left: Violin plots represent miR-183-5p expression in Cancerous versus Normal breast tissue ( p -value = 3.35 × 10 −21 ) and in Luminal A versus TNBC ( p -value = 0.25). Right: Violin plots depicting PLK1 expression in Cancerous versus Normal breast tissue ( p -value = 4.22 × 10 −20 ) and in Luminal A versus TNBC. ( p -value = 1.01 × 10 −6 ). N = 46 paired luminal A, 15 paired TNBC, 61 cancer vs normal.

Article Snippet: To confirm that miR-183-5p binding was specifically responsible for the reduction of PLK1 protein, PLK1 3′UTR was inserted downstream of the PLK1 CDS expression vector (Origene, Rockville, MD, USA), generating PLK1 CDS-only and PLK1 CDS + 3′UTR expression vectors.

Techniques: Luciferase, Activity Assay, Transfection, Negative Control, Western Blot, Expressing, Quantitative RT-PCR, Control

Journal: Cell Death and Differentiation

Article Title: Synergistic apoptotic effect of miR-183-5p and Polo-Like kinase 1 inhibitor NMS-P937 in breast cancer cells

doi: 10.1038/s41418-021-00864-2

Figure Lengend Snippet: A WST8 assay demonstrating the effects of NMS-P937 treatment: 0, 10, 25, 50, 75, 100, and 150 nM after transfection with negative control (NC) or miR-183-5p or si-PLK1. Left hand graphs. TNBC cell lines MDA-MB-231 (top left, p -value = 3.26e-14) and BT549 (top right, p -value = 0.00112). Luminal A cell lines T47D (lower left, p -value < 2e-16) and ZR-75-1 (lower right p -value = 1.93e-6). Right hand graphs. Assay was repeated using si-PLK1. Data ( N = 3/group) are mean + SD. ** p -value <0.05, *** p -value <0.001 calculated by ANOVA. B Left: Matrices demonstrating MDA-MB-231 and T47D cell lines treated with different concentrations of miR-183-5p and NMS-P937, then analyzed to determine cell mortality (% inhibition). Right: Bliss score shows a synergistic effect present on cell mortality between miR-183-5p overexpression and NMS-P937 treatment.

Article Snippet: To confirm that miR-183-5p binding was specifically responsible for the reduction of PLK1 protein, PLK1 3′UTR was inserted downstream of the PLK1 CDS expression vector (Origene, Rockville, MD, USA), generating PLK1 CDS-only and PLK1 CDS + 3′UTR expression vectors.

Techniques: Transfection, Negative Control, Inhibition, Over Expression

Journal: Cell Death and Differentiation

Article Title: Synergistic apoptotic effect of miR-183-5p and Polo-Like kinase 1 inhibitor NMS-P937 in breast cancer cells

doi: 10.1038/s41418-021-00864-2

Figure Lengend Snippet: A MDA-MB-231, BT549, T47D, and ZR-75-1 cells treated with NMS-P937 for 48 h, +/- miR-183-5p transfection for 72 h. Total protein lysates were analyzed by Western blot. Blots were stained for PLK1, DNMT1, p-STAT3 (Tyr 705), p-p53 (Ser15), Bcl-XL, cleaved caspases 7 and 3, cleaved PARP and vinculin. B – E Protein band densitometry normalized to vinculin. Data ( N = 3/group) are presented as mean + SD.

Article Snippet: To confirm that miR-183-5p binding was specifically responsible for the reduction of PLK1 protein, PLK1 3′UTR was inserted downstream of the PLK1 CDS expression vector (Origene, Rockville, MD, USA), generating PLK1 CDS-only and PLK1 CDS + 3′UTR expression vectors.

Techniques: Transfection, Western Blot, Staining

Journal: Cell Death and Differentiation

Article Title: Synergistic apoptotic effect of miR-183-5p and Polo-Like kinase 1 inhibitor NMS-P937 in breast cancer cells

doi: 10.1038/s41418-021-00864-2

Figure Lengend Snippet: A MDA-MB-231, BT549, T47D, and ZR-75-1 cells treated with NMS-P937 for 48 h, +/- si-PLK1 for 72 h. Total protein lysates were analyzed by Western blot. Blots were stained for PLK1, DNMT1, p-STAT3 (Tyr 705), p-p53 (Ser15), Bcl-XL, cleaved caspases 7 and 3, cleaved PARP and vinculin. B – E Protein band densitometry normalized to vinculin. Data ( N = 3/group) are presented as mean + SD.

Article Snippet: To confirm that miR-183-5p binding was specifically responsible for the reduction of PLK1 protein, PLK1 3′UTR was inserted downstream of the PLK1 CDS expression vector (Origene, Rockville, MD, USA), generating PLK1 CDS-only and PLK1 CDS + 3′UTR expression vectors.

Techniques: Western Blot, Staining

Journal: Cell Death and Differentiation

Article Title: Synergistic apoptotic effect of miR-183-5p and Polo-Like kinase 1 inhibitor NMS-P937 in breast cancer cells

doi: 10.1038/s41418-021-00864-2

Figure Lengend Snippet: A Representative flow cytometry images of breast cancer cell lines treated with NMS-P937 for 48 h, +/- si-PLK1 transfection for 72 h. Cells were stained with Annexin-FITC and PI. The cell populations of interest were those undergoing Late (Q2) or early (Q4) apoptosis. B , C Bar graphs representing the percentage of cells undergoing early (B) or late (C) apoptosis. Cells were either treated with negative control miRNA (NC), si-PLK1 only, NMS-P937 only (25 nM or 75 nM), or si-PLK1 + NMS-P937. Data ( N = 4/group) are presented as mean + SD. * p -value <0.05, ** p -value <0.001 compared to negative control by Student’s unpaired t test.

Article Snippet: To confirm that miR-183-5p binding was specifically responsible for the reduction of PLK1 protein, PLK1 3′UTR was inserted downstream of the PLK1 CDS expression vector (Origene, Rockville, MD, USA), generating PLK1 CDS-only and PLK1 CDS + 3′UTR expression vectors.

Techniques: Flow Cytometry, Transfection, Staining, Negative Control