Journal: Cancer medicine

Article Title: Arid4a Suppresses Breast Tumor Metastasis by Enhancing MTSS1 Expression via mRNA Stability.

doi: 10.1002/cam4.70732

Figure Lengend Snippet: FIGURE 1 | Arid4a is expressed at relatively low levels in breast tumors and indicates poor prognosis. (A) The expression of Arid4a between nor- mal breast tissues and primary breast tumors was analyzed (http://ualcan.path.uab.edu/analysis-prot.html). (B) Arid4a expression was measured by qPCR in human breast tumors and surrounding healthy tissues (n = 16). (C) Comparison of Arid4a expression between different subtypes of breast cancers (http://ualcan.path.uab.edu/analysis-prot.html). (D) Arid4a protein expression in human primary breast tumors and normal tissues was analyzed (http://ualcan.path.uab.edu/analysis-prot.html). (E) Left: Representative images of Arid4a IHC stating in normal mammary gland tissues and breast tumor tissues were shown. Scale bar, 100 μm. Right: IHC scores of Arid4a staining. (F) Arid4a protein levels were measured by immunoblotting in human normal mammary gland epithelial cell lines (MCF-10A and MCF-12A) and breast tumor cell lines (MDA-MB-231, MCF7, MDA-MB-468, and T47D). (G) Arid4a expression was analyzed based on nodal metastasis status of breast cancers (http://ualcan.path.uab.edu/ analysis-prot.html). (H) Kaplan–Meier overall survival and relapse free survival curves of breast cancer patients with low and high tumor Arid4a transcripts (kmplot.com). (I) Kaplan–Meier distant metastasis free survival (DMFS), disease free survival (DFS), relapse free survival, and disease specific survival (DSS) curves of breast cancer patients with low and high tumor Arid4a transcripts (PrognoScan: A new database for meta-analysis of the prognostic value of genes. (kyutech.ac.jp)). *p < 0.05; ****p < 0.00001.

Article Snippet: Human breast cancer cell lines (MDA- MB- 231, MDA- MB- 468, MCF7 and T47D) and human normal mammary epithelial cell lines (MCF- 10A and MCF- 12A) were purchased from the American Type Culture Collection (ATCC).

Techniques: Expressing, Comparison, Staining, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: The Antioxidant Transcription Factor Nrf2 Negatively Regulates Autophagy and Growth Arrest Induced by the Anticancer Redox Agent Mitoquinone *

doi: 10.1074/jbc.M110.133579

Figure Lengend Snippet: Growth inhibition and cell cycle arrest by MitoQ or CoQ10. A and B, an SRB dye-based assay was used for measuring cell viability following increasing concentrations of either MitoQ (left) or CoQ10 (right) after 72 h. Breast cancer cell lines (MDA-MB-231 or MCF-7) or healthy breast epithelial cells (MCF-12A) were treated as indicated. B, a colony formation assay was performed on MDA-MB-231 cells incubated with MitoQ. Cells were incubated with MitoQ for 6 h, followed by replacement of medium and replating for a colony formation period of 7 days. C, MDA-MB-231 cells were treated with either MitoQ or CoQ10, as indicated, for 48 h. The cells were fixed and stained with PI followed by flow cytometric analysis. The percentage of cells in each cell cycle phase was quantitated and is provided in each panel. D, the phosphorylation of Chk1 and Chk2 kinases was assayed using confocal microscopy. Representative images of increased nuclear foci corresponding to phospho-Chk1-Ser317 (pChk1-S317), phospho-Chk2-Thr68 (pChk1-T68), and cytoplasmic phospho-Chk1-Ser345 (pChk1-S345) after 0.1 μm MitoQ or 0.1 μm doxorubicin (Dox) treatment (24 h) are shown. Error bars, S.D.

Article Snippet: MCF-12A healthy human mammary epithelial cells were obtained from BioWhittaker Clonetics (Walkersville, MD) and maintained in mammary epithelial cell growth medium ( 16 ).

Techniques: Inhibition, Colony Assay, Incubation, Staining, Confocal Microscopy

Journal: Journal of Cell Science

Article Title: Centrosome amplification promotes cell invasion via cell–cell contact disruption and Rap-1 activation

doi: 10.1242/jcs.261150

Figure Lengend Snippet: CA increases in vitro and in vivo migration and invasion, and blocking CA diminishes the metastatic characteristics of MDA-MB-231 cells. (A) Representative images of immunofluorescently stained −CA and +CA MCF10A cells fixed with methanol/5 mM EGTA solution and stained for MTs (α-tubulin, red), centrosomes (γ-tubulin, green) and counterstained with DAPI (DNA, blue). The +CA panels show a multipolar mitotic (mitotic catastrophe) orchestrated by amplified centrosomes and clustered centrosomes in a pseudobipolar mitotic. The −CA panel shows a cell with a normal centrosome complement of 2. Scale bar: 10 μm. Images are representative of n =3 independent experiments. (B) Fluorescence microscopy quantification of the percentage of +CA cells at each time point post-Dox treatment; normal centrosome number (≤2 per cell) and amplified centrosomes or CA (≥3 per cell). Bar graphs represent mean±s.e.m. from three independent experiments; ≥200 cells/time point. (C,D) Transwell invasion (C) and migration (D) assay results showing that CA induction significantly increases cellular invasion and migration in MCF10A PLK4 cells compared to controls −CA PLK4 and DOX+ PLK4 1-608 . The bar graph represents the mean±s.e.m. number of cells invaded and migrated from four independent experiments. * P ≤0.05; ** P ≤0.01; ns, not significant (one-way ANOVA and Bonferroni's multiple comparison test). (E) Representative images from four independent experiments for the chicken xenograft assay for the MCF10A −CA and +CA cell–Matrigel graft. Images show H&E staining (upper panels; Matrigel staining is pink) and immunohistochemical staining for cytokeratin 8 (Cy8; lower panels) on formalin-fixed paraffin-embedded tissue sections −CA and +CA MCF10A PLK4 cells–Matrigel grafts. The +CA graft shows moderate to strong chicken-graft focal reaction (yellow arrow), with multifocal heterophil infiltration in the intermediate mesoderm layer (yellow circles), invasion of epithelial cell nests into chicken mesodermal layer (black arrows and circles) and dilated lymphatics in the intermediate mesoderm layer (green arrows). Mild to moderate focal reaction of the CAM to the graft was noted in −CA grafts. The corresponding IHC (lower panels) shows that the invaded epithelial cell nests were positive for Cy8 (dotted circles). Scale bars: 500 µm (top and bottom panels); 250 µm (middle panels). (F,G) Transwell invasion (F) and migration assays (G) show that blocking endogenous CA with 150 nM Centrinone B for 16 h in the TNBC cell line MDA-MB-23 significantly decreased cellular invasion and migration. Migrated or invaded cells were quantified with DAPI staining and the cells counted using immunofluorescence images from five different fields at 20× magnification. The box represents the 25–75th percentiles, and the median is indicated. The whiskers show the data spread from two independent biological repeats. * P ≤0.05, *** P <0.001 (unpaired two-tailed Student's t -test). WT, wild-type.

Article Snippet: The breast cancer cell lines MDA-MB-231, MDA-MB-468 (ATCC) and doxycycline-inducible MCF7 PLK4 cells (a gift from Susana Godinho; ) were cultured in RPMI-1640 (Sigma R8758) supplemented with 10% fetal bovine serum (Sigma F7524) and 1% penicillin-streptomycin (Sigma P4333).

Techniques: In Vitro, In Vivo, Migration, Blocking Assay, Staining, Amplification, Fluorescence, Microscopy, Comparison, Xenograft Assay, Immunohistochemical staining, Formalin-fixed Paraffin-Embedded, Immunofluorescence, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Centrosome amplification promotes cell invasion via cell–cell contact disruption and Rap-1 activation

doi: 10.1242/jcs.261150

Figure Lengend Snippet: CA-induced migration, invasion and cell–ECM attachment involves activation of Rap1. (A) Western blot of Rap1 pulldown assay in MCF10A PLK4 showing CA induced increased GTP-bound Rap1. Negative (GDP) and positive (GTPγS) controls on untreated cell lysates were included. Ponseau stain is included as a loading control for the pulldown. Total Rap1 in 60 µg of total cell lysates is shown in the bottom panel. The image shown is representative of n =3 independent repeats and the fold-change represents the quantification of +CA and −CA bands by densitometry. (B) CA increases MCF10A cell–ECM adherence in a Rap1-dependent manner. Cell adhesion assays was performed on MCF10A PLK4 cells 48 h post-Dox (2 μg/ml) or without Dox, and 3 h treatment with or without 10 µM GGTI-298. Cells bound to the matrix were quantified with Crystal Violet staining; five 20× fields per well were counted and the bar graph represents mean±s.e.m. from four independent experiments. ** P ≤0.01; ns, not significant (two-way ANOVA and Bonferroni's multiple comparison test). (C,D) Rap1 inhibition significantly decreased CA-induced cellular invasion and migration. The bar graphs represent mean±s.e.m. number of cells invaded and migrated, quantified by Crystal Violet staining in five random 20× fields per insert from four independent experiments. * P ≤0.05; ** P ≤0.01; ns, not significant (two-way ANOVA and Bonferroni's multiple comparison test). (E) Representative H&E images of the chicken xenograft assay with cell–Matrigel graft. MCF10A PLK4 cells were xenografted 48 h post-Dox (2 μg/ml) or without Dox treatment and 3 h treatment with or without 10 µM GGTI-298. Rap1 inhibition decreased the CA-induced bidirectional chicken-graft focal reaction in vivo . Scale bars: 500 µm. (F) Area (mm 2 ) of chicken-graft focal reaction was quantified in scans of MCF10A grafts in +CA or –CA with or without GGTI-298 using Olympus OlyVIA software. Bar graphs represent mean±s.e.m. from three independent experiments. *** P ≤0.001; ns, not significant (two-way ANOVA and Bonferroni's multiple comparison test). WT, wild-type.

Article Snippet: The breast cancer cell lines MDA-MB-231, MDA-MB-468 (ATCC) and doxycycline-inducible MCF7 PLK4 cells (a gift from Susana Godinho; ) were cultured in RPMI-1640 (Sigma R8758) supplemented with 10% fetal bovine serum (Sigma F7524) and 1% penicillin-streptomycin (Sigma P4333).

Techniques: Migration, Activation Assay, Western Blot, Staining, Control, Comparison, Inhibition, Xenograft Assay, In Vivo, Software

Journal: Journal of Cell Science

Article Title: Centrosome amplification promotes cell invasion via cell–cell contact disruption and Rap-1 activation

doi: 10.1242/jcs.261150

Figure Lengend Snippet: Long-term MCF10A PLK4 wild-type cell culture Transwell system. (A) Experimental layout of long-term Transwell membrane cell culture system. (B) Representative immunofluorescence images from four experiments of MCF10A PLK4 wild-type cells cultured for 47 days. CA was induced by treating at the specified intervals with 2 µg/ml Dox. The cells were fixed and permeabilised in 3.7% PFA and methanol/EGTA. Centrosomes were labelled for γ-tubulin (green) and AJs with β-catenin (red), and DNA counterstained with DAPI. Disrupted and mislocalised β-catenin are seen in +CA cells compared to –CA cells showing well-formed β-catenin at the apical region. Scale bar: 20 μm. (C) The bar graph shows a significant increase in percentage CA within the apical and middle layers of the multi-layered long-term culture. Bar graphs represent mean±s.e.m. from three biological repeats. *** P ≤0.001 (unpaired two-tailed Student's t -test). (D) TEM images of sagittal view of the multi-layered cell structure with altered intercellular space in response to CA induction. Images representative of four independent experiments. Scale bars: 4 µm. (E) XY and Z -stack projections showing disruption of the TJ protein ZO-1 (green) and the AJ protein β-catenin (red) at the apical side in +CA conditions. Localisation of both ZO-1 and β-catenin was found to be primarily within the apical region rather than the middle or basal regions in long-term culture system. Scale bar: 20 μm.

Article Snippet: The breast cancer cell lines MDA-MB-231, MDA-MB-468 (ATCC) and doxycycline-inducible MCF7 PLK4 cells (a gift from Susana Godinho; ) were cultured in RPMI-1640 (Sigma R8758) supplemented with 10% fetal bovine serum (Sigma F7524) and 1% penicillin-streptomycin (Sigma P4333).

Techniques: Cell Culture, Membrane, Immunofluorescence, Two Tailed Test, Disruption

Journal: Journal of Cell Science

Article Title: Centrosome amplification promotes cell invasion via cell–cell contact disruption and Rap-1 activation

doi: 10.1242/jcs.261150

Figure Lengend Snippet: CA disrupts apical junctional complexes in MCF10A PLK4 cells. (A) Representative confocal images of cells co-stained for (left panels) the AJ protein β-catenin (red) and TJ protein ZO-1 (green), and (right panels) TJ proteins JAM-A (red), occludin (green) showing localisation and intactness of epithelial cell junctions in +CA and –CA MCF10A PLK4 cells. The cells were fixed in 3.7% PFA and permeabilised in 0.5% Triton X-100. Images are representative of three biological repeats. Scale bars: 20 μm. (B,C) Graphs represent mean±s.e.m. percentage cell junction staining disruption in 10 fields of view/slide (B) and average grey value intensity values over 10 fields of view (C). CA significantly disrupted cell junctions and altered the protein localisation. ** P ≤0.01, *** P <0.001 (paired two-tailed Student's t -test). (D) Representative TEM images of +CA and –CA TJs, AJs and DSs indicated by asterisk (*) taken at 20,000×, HV=100.0 kV. Scale bars: 600 nm. (E) Bar graph represents the cell junction quantification showing a significant downregulation of TJs and AJs (in apical region) and no significant difference in desmosomes due to CA. Bars represent mean±s.e.m. junction number from four independent experiments (10 images per experiment). * P <0.05, ** P ≤0.01; ns, not significant (unpaired two-tailed Student's t -test).

Article Snippet: The breast cancer cell lines MDA-MB-231, MDA-MB-468 (ATCC) and doxycycline-inducible MCF7 PLK4 cells (a gift from Susana Godinho; ) were cultured in RPMI-1640 (Sigma R8758) supplemented with 10% fetal bovine serum (Sigma F7524) and 1% penicillin-streptomycin (Sigma P4333).

Techniques: Staining, Disruption, Two Tailed Test

Journal: Journal of Cell Science

Article Title: Centrosome amplification promotes cell invasion via cell–cell contact disruption and Rap-1 activation

doi: 10.1242/jcs.261150

Figure Lengend Snippet: CA disrupts epithelial cell–cell adhesion and increases active Rap1 levels in tumorigenic breast cancer cells. (A) Experimental layout of a long-term MCF7 PLK4 cell culture system. (B) Bar graph shows percentage of +CA MCF7 PLK4 cells at each time point post-Dox (2 μg/ml). Cells were fixed with methanol/5 mM EGTA solution and stained for centrosomes (γ-tubulin, green) and MTs (α-tubulin, red), and counterstained with DAPI (DNA, blue). Bars represent mean±s.e.m., n =3; ≥200 cells/time point. (C) Representative images of +CA and –CA MCF7 cells co-stained for the AJ protein β-catenin (red), the TJ protein ZO-1 (green) (top panels), and the TJ proteins JAM-A (red) and occludin (green) (bottom panels). Scale bars: 20 μm. (D) Bar graph representing mean±s.e.m. the percentage cell junction staining disruption in 10 fields of view/slide. (E) Representative TEM images of MCF7 PLK4 cells showing cell junctions in +CA and –CA cells (indicated by asterisk). Cells were imaged at a magnification of 20,000×, HV=100.0 kV. Scale bars: 600 nm. (F) Bar graph represents cell junction quantification for +CA and –CA, with a clear decrease in TJ formation in +CA MCF7 PLK4 cells. Bars represent mean±s.e.m. cell junction number from four independent experiments. No significance was found (two-way ANOVA with Bonferroni post-test). (G) Western blot of Rap1 pulldown assay in MCF7 PLK4 cells showing –CA cells had increased GTP-bound Rap1. Negative (GDP) and positive (GTPγS) controls on untreated cell lysates were included. Ponseau stain is included as a loading control for the pulldown. Total Rap-1 in 60 µg of total cell lysates is shown in the bottom panel. The image shown is representative of n =3 independent repeats and the fold-change +CA represents the quantification of ±CA bands by densitometry.

Article Snippet: The breast cancer cell lines MDA-MB-231, MDA-MB-468 (ATCC) and doxycycline-inducible MCF7 PLK4 cells (a gift from Susana Godinho; ) were cultured in RPMI-1640 (Sigma R8758) supplemented with 10% fetal bovine serum (Sigma F7524) and 1% penicillin-streptomycin (Sigma P4333).

Techniques: Cell Culture, Staining, Disruption, Western Blot, Control