Journal: Cell Reports Medicine

Article Title: Hemagglutinin stalk-binding antibodies enhance effectiveness of neuraminidase inhibitors against influenza via Fc-dependent effector functions

doi: 10.1016/j.xcrm.2022.100718

Figure Lengend Snippet:

Article Snippet: 16 hours after infection, the media was replaced with 50 μL of assay buffer (RPMI 1640 supplemented with 4% (vol/vol) low IgG FBS(Gibco)) containing oseltamivir carboxylate (Toronto Research Chemicals) or baloxavir marboxil (Selleck Chemicals) and serial dilutions of antibody or serum.

Techniques: Control, Virus, Recombinant, Construct, Cell Culture, Expressing, Plasmid Preparation, Bioassay, Variant Assay, Luciferase, Software

Journal: iScience

Article Title: Immunometabolic adaptation in monocytes underpins functional changes during pregnancy

doi: 10.1016/j.isci.2024.109779

Figure Lengend Snippet:

Article Snippet: CD163 human antibody (clone GHI/61.1) , Miltenyi , Cat#130-123-249; RRID: AB_2819455.

Techniques: Recombinant, Adhesive, Sequencing, Modification, DC Protein Assay, Staining, Imaging, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Gene Expression, Software, Saline

Journal: eLife

Article Title: NuRD and CAF-1-mediated silencing of the D4Z4 array is modulated by DUX4-induced MBD3L proteins

doi: 10.7554/eLife.31023

Figure Lengend Snippet: ( A–C ) DUX4 and DUX4 target gene expression as determined by RT-qPCR in MB2401 control ( A ), MB073 FSHD1 ( B ) or MB200 FSHD2 ( C ) myoblasts without (-) or with (+) doxycycline (Dox) treatment for 48 hr to induce MBD3L2 transgene expression in clonal cell lines. ( D–E ) DUX4-positive nuclei upon overexpression of MBD3L2 in MB200 FSHD2 myoblasts as in ( C ) were detected by immunofluorescence ( D ) and quantified by counting three fields representing >125 nuclei ( E ). ( F–G ) DUX4 and DUX4 target gene expression as determined by RT-qPCR following control (CTRL) or MBD3L family gene shRNA knockdown in MB073 FSHD1 ( F ) or MB200 FSHD2 ( G ) myotubes. Error bars denote the standard deviation from the mean of three biological replicates. Statistical significance was calculated by comparing the specific knockdown to the control knockdown for each gene using a two-tailed, two-sample Mann-Whitney U test and p was ≤0.05 for all comparisons except in ( A ). See also . 10.7554/eLife.31023.038 Figure 6—source data 1. Source data for . This file contains the source data used to make the graphs presented in and – . GraphPad Prism was utilized to visually represent the quantitative data.

Article Snippet: Recombinant DNA reagent , pCW57.1-MBD3L2 , This paper , Addgene plasmid #106332 , Lentiviral vector expressing doxycycline-inducible MBD3L2 (generated using pCW57.1 [Addgene plasmid #41393]).

Techniques: Expressing, Quantitative RT-PCR, Over Expression, Immunofluorescence, shRNA, Standard Deviation, Two Tailed Test, MANN-WHITNEY

Journal: eLife

Article Title: NuRD and CAF-1-mediated silencing of the D4Z4 array is modulated by DUX4-induced MBD3L proteins

doi: 10.7554/eLife.31023

Figure Lengend Snippet: ( A–E ) The ectopic ( A–C ) or endogenous ( D–E ) expression of MBD3L2 as determined by RT-qPCR in MB2401 control ( A ), MB073 FSHD1 ( B ) or MB200 FSHD2 ( C ) myoblasts cultured without (-) or with (+) doxycycline (Dox) for 48 hr, or in MB073 FSHD1 ( D ) or MB200 FSHD2 ( E ) myotubes expressing control (CTRL) or MBD3L gene shRNAs. Error bars denote the standard deviation from the mean of three biological replicates. See also .

Article Snippet: Recombinant DNA reagent , pCW57.1-MBD3L2 , This paper , Addgene plasmid #106332 , Lentiviral vector expressing doxycycline-inducible MBD3L2 (generated using pCW57.1 [Addgene plasmid #41393]).

Techniques: Expressing, Quantitative RT-PCR, Cell Culture, Standard Deviation

Journal: eLife

Article Title: NuRD and CAF-1-mediated silencing of the D4Z4 array is modulated by DUX4-induced MBD3L proteins

doi: 10.7554/eLife.31023

Figure Lengend Snippet: ( A–D ) MBD3L2, DUX4, and DUX4 target gene expression as determined by RT-qPCR in two additional independent experiments with control (CTRL) or MBD3L shRNA-expressing MB073 FSHD1 ( A–B ) or MB200 FSHD2 ( C–D ) muscle cell lines differentiated into myotubes. Error bars denote the standard deviation from the mean of three biological replicates. See also .

Article Snippet: Recombinant DNA reagent , pCW57.1-MBD3L2 , This paper , Addgene plasmid #106332 , Lentiviral vector expressing doxycycline-inducible MBD3L2 (generated using pCW57.1 [Addgene plasmid #41393]).

Techniques: Expressing, Quantitative RT-PCR, shRNA, Standard Deviation

Journal: eLife

Article Title: NuRD and CAF-1-mediated silencing of the D4Z4 array is modulated by DUX4-induced MBD3L proteins

doi: 10.7554/eLife.31023

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pCW57.1-MBD3L2 , This paper , Addgene plasmid #106332 , Lentiviral vector expressing doxycycline-inducible MBD3L2 (generated using pCW57.1 [Addgene plasmid #41393]).

Techniques: Recombinant, Plasmid Preparation, Expressing, Generated, Modification, Knock-Out, 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: 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

Journal: Journal of Virology

Article Title: Merkel Cell Polyomavirus Small T Antigen Drives Cell Motility via Rho-GTPase-Induced Filopodium Formation

doi: 10.1128/JVI.00940-17

Figure Lengend Snippet: MCPyV ST expression results in the upregulation of several actin-associated proteins. (A) i293-ST cells remained uninduced or were incubated for 48 h in the presence of doxycycline hyclate (Dox) (i) or MCC13 cells were transfected with 1 μg EGFP or EGFP-ST for 12 h (ii). The cell lysates were then probed with Arp3-, cortactin-, and cofilin-specific antibodies. GAPDH was used as a measure of equal loading, and the 2T2 hybridoma was used to confirm MCPyV ST expression. (B) Densitometry quantification of the Western blots was carried out using Image J software and is shown as a percentage relative to the loading control, GAPDH ( n = 3). (C) Total RNA was extracted from uninduced (Uni) or induced (In) i293-ST cells after 24 h, and relative transcript levels were analyzed by RT-qPCR using GAPDH as a reference. The fold increase was determined by ΔΔ C T , and statistical significance was analyzed using a nonpaired t test. Data from 3 independent experiments are presented as the fold increase versus uninduced control. *, P < 0.001. (D) FFPE sections of two primary MCC tumors were stained with CK20-, MCPyV LT-, and cortactin-specific antibodies or an isotype negative control. The sections were then incubated with Alexa Fluor-labeled secondary antibodies and analyzed using a Zeiss LSM 510 confocal laser scanning microscope. (E) Immunoblot analysis was performed on the cellular lysates of two independent MCC tumor samples and a negative-control nontumor cadaveric skin sample using Arp3- and cortactin-specific antibodies. GAPDH was used as a measure of equal loading, and the 2T2 hybridoma was used to confirm MCPyV ST expression. (F) Densitometry quantification of the Western blots was carried out using Image J software and is shown as a percentage relative to the loading control, GAPDH. The data were analyzed using three replicates per experiment ( n = 3), and statistical analysis was done with a two-tailed t test with unequal variance. *, P < 0.01. (G) The MCPyV-positive MCC cell line WAGA was transduced with lentivirus expressing a scrambled shRNA or an ST-targeting shRNA. Upon ST depletion, the cell lysates were probed with Arp3- and cortactin-specific antibodies. GAPDH was used as a measure of equal loading, and the 2T2 hybridoma was used to confirm MCPyV ST expression. (H) Densitometry quantification of the Western blots was carried out using Image J software and is shown as a percentage relative to the loading control, GAPDH ( n = 3). The error bars indicate standard deviations.

Article Snippet: ST-Flag, EGFP, and EGFP-ST expression was induced from i293-ST , i293-GFP, and i293-GFP-ST cells, respectively, with 2 μg/ml doxycycline hyclate for up to 48 h. The cells were plated in 6-well plates, and transfections routinely used 1 μg plasmid DNA and Lipofectamine 2000 (Life Technologies) or 5 μg plasmid DNA and nucleofection (Lonza), following the manufacturers' instructions.

Techniques: Expressing, Incubation, Transfection, Western Blot, Software, Control, Quantitative RT-PCR, Staining, Negative Control, Labeling, Laser-Scanning Microscopy, Two Tailed Test, Transduction, shRNA

Journal: Journal of Virology

Article Title: Merkel Cell Polyomavirus Small T Antigen Drives Cell Motility via Rho-GTPase-Induced Filopodium Formation

doi: 10.1128/JVI.00940-17

Figure Lengend Snippet: The integrin inhibitor RGDS reduces MCPyV ST-induced cell motility and filopodium formation. (A) i293-GFP or i293-GFP-ST cells were transfected with 1 μg HA-Cdc42 or HA-Rac1 and then induced with doxycycline hyclate for 6 h. The cell lysates were probed for phosphorylated Cdc42/Rac1 at the S71 residue. HA tag- and GAPDH-specific antibodies were used to measure equal loading. 2T2 was used to probe for MCPyV ST expression. (B and D) i293-EGFP and i293-EGFP-ST cells were induced using doxycycline hyclate (B), or MCC13 cells were transfected with 1 μg of EGFP and EGFP-ST (D). After 24 h, the cells were treated with 1 μg/μl DMSO or 1 μM, 10 μM, 50 μM, or 100 μM RGDS. After 24 h, cell motility was analyzed using an IncuCyte kinetic live-cell-imaging system. Images were taken every 30 min over a 24-h period. The movements of the cells were then tracked using ImageJ software. (C and E) The average distance traveled was measured ( n = 25 per condition), and significance was tested using a 3-tailed Student t test. *, P < 0.001. (F) HEK-293 cells were transfected with 1 μg EGFP or EGFP-ST and then treated with 100 μM RGDS. After 24 h, the cells were fixed, and GFP fluorescence was analyzed by direct visualization; in addition, the cells were stained with rhodamine-phalloidin. (G) The number and length of actin-based protrusions were analyzed for 50 cells per condition using ImageJ software. The error bars indicate standard deviations.

Article Snippet: ST-Flag, EGFP, and EGFP-ST expression was induced from i293-ST , i293-GFP, and i293-GFP-ST cells, respectively, with 2 μg/ml doxycycline hyclate for up to 48 h. The cells were plated in 6-well plates, and transfections routinely used 1 μg plasmid DNA and Lipofectamine 2000 (Life Technologies) or 5 μg plasmid DNA and nucleofection (Lonza), following the manufacturers' instructions.

Techniques: Transfection, Residue, Expressing, Live Cell Imaging, Software, Fluorescence, Staining