Journal: eLife

Article Title: Computational and cellular studies reveal structural destabilization and degradation of MLH1 variants in Lynch syndrome

doi: 10.7554/eLife.49138

Figure Lengend Snippet: ( A ) Example of the immunofluorescence imaging of HCT116 cells using antibodies to MLH1. Hoechst staining was used to mark the nucleus. Note the reduced steady-state levels of the G67R MLH1 variant compared to wild-type MLH1. ( B ) The total fluorescent intensity for each of the 69 different MLH1 variants was determined after excluding the non-transfected cells and normalizing the intensities to that for wild-type MLH1. The intensities were then plotted vs. the predicted ΔΔG values. Between 200 and 1,000 cells were included for each quantification. The error bars indicate the standard error of the mean (n = 5 experiments). Each variant is color-coded according to the ClinVar disease category. ( C ) Distribution of steady-state levels by DME category – 0 is loss-of-function in all assays by , 3 represents function in all these assays (for details see the Materials and Methods). Raincloud plot visualization as described in . Colored surface, smoothed density estimate. Gray dots represent means within each DME category, with bars for standard error. ( D ) Distribution of FoldX ΔΔGs across DME categories (as in ( C )). ( E ) FoldX ΔΔGs for all variants tested in this work, indicating their position in the MLH1 sequence. As elsewhere, values above 15 kcal/mol were here set to this value.

Article Snippet: Cell line ( Homo sapiens ) , HCT116 , ATCC , CCL-247EMT; RRID: CVCL_0291 , -.

Techniques: Immunofluorescence, Imaging, Staining, Variant Assay, Transfection, Sequencing

Journal: eLife

Article Title: Computational and cellular studies reveal structural destabilization and degradation of MLH1 variants in Lynch syndrome

doi: 10.7554/eLife.49138

Figure Lengend Snippet: ( A ) HCT116 cells transfected with the indicated variants were analyzed by blotting with antibodies to MLH1. Co-transfection with a plasmid expressing GFP was included to test the transfection efficiencies between the MLH1 variants. β-actin served as a loading control. ( B ) Quantification of blots as in ( A ) normalized to the steady-state level of wild-type (WT) MLH1. The error bars show the standard deviation (n = 3). ( C ) MLH1-transfected HCT116 cells were treated with 25 µg/mL cycloheximide (CHX) for 0, 4, 8 or 12 hr, and lysates were analyzed by blotting using antibodies to MLH1. β-actin was used as a loading control. ( D ) Quantification of blots as in panel ( C ), normalized to the steady-state levels at t = 0 hr. The error bars indicate the standard deviation (n = 3). ( E ) Western blotting with antibodies to MLH1 of whole cell lysates from transfected cells either untreated or treated for 16 hr with 10 µM bortezomib (BZ). Blotting for β-actin was included as a loading control.

Article Snippet: Cell line ( Homo sapiens ) , HCT116 , ATCC , CCL-247EMT; RRID: CVCL_0291 , -.

Techniques: Transfection, Cotransfection, Plasmid Preparation, Expressing, Control, Standard Deviation, Western Blot

Journal: eLife

Article Title: Computational and cellular studies reveal structural destabilization and degradation of MLH1 variants in Lynch syndrome

doi: 10.7554/eLife.49138

Figure Lengend Snippet: ( A ) The levels of endogenous PMS1 and PMS2 were determined by blotting of whole-cell lysates of HCT116 cells transfected with either empty vector or with wild-type MLH1 and treated with 25 µg/mL cycloheximide (CHX) for 0, 4, 8 or 12 hr. The antibodies used were to PMS1 and PMS2, and as a control to MLH1. β-actin served as loading control. ( B ) Quantification of blots as in panel ( A ) normalized to protein levels at 0 hr. The error bars indicate the standard deviation (n = 3). ( C ) The levels of endogenous MLH1, PMS1 and PMS2 were compared by blotting of cell lysates of HCT116 cells either untreated, or treated with cycloheximide (CHX) or with bortezomib (BZ) and CHX. β-actin served as loading control. ( D ) The levels of endogenous PMS1 and PMS2 and transfected MLH1 were compared by western blotting using antibodies to PMS1, PMS2 and MLH1. β-actin served as loading control. ( E ) Quantification of blots as in panel ( C ) normalized to the level of endogenous PMS1 (grey) or PMS2 (red) in untransfected HCT116 cells. The error bars show the standard deviation (n = 3). ( F ) Plotting the levels of the MLH1 variants vs. the levels of endogenous PMS1 (grey) and PMS2 (red). The error bars show the standard deviation (n = 3). ( G ) The levels of MLH1 and YFP-tagged PMS2 were analyzed by SDS-PAGE and blotting of whole-cell lysates of HCT116 cells transfected with the indicated expression plasmids. β-actin was included as loading control. ( H ) Co-transfected PMS2-YFP was immunoprecipitated (IP) using GFP-trap beads, and the precipitated material was analyzed by electrophoresis and blotting. Bortezomib was added to all cultures 16 hr prior to cell lysis to ensure ample amounts of the unstable MLH1 variants.

Article Snippet: Cell line ( Homo sapiens ) , HCT116 , ATCC , CCL-247EMT; RRID: CVCL_0291 , -.

Techniques: Transfection, Plasmid Preparation, Control, Standard Deviation, Western Blot, SDS Page, Expressing, Immunoprecipitation, Electrophoresis, Lysis

Journal: eLife

Article Title: Computational and cellular studies reveal structural destabilization and degradation of MLH1 variants in Lynch syndrome

doi: 10.7554/eLife.49138

Figure Lengend Snippet:

Article Snippet: Cell line ( Homo sapiens ) , HCT116 , ATCC , CCL-247EMT; RRID: CVCL_0291 , -.

Techniques: Recombinant, Software

Journal: The FASEB Journal

Article Title: Vimentin‐mediated myosin 10 aggregation at tips of cell extensions drives MT1‐MMP‐dependent collagen degradation in colorectal cancer

doi: 10.1096/fj.202300672r

Figure Lengend Snippet: FIGURE 3 Vimentin regulates myosin10 substitution dynamics. (A) Representative live cell images for FRAP in SW480 cells transfected with GFP-Myo10. Images before bleaching (Pre-bleach), after bleach (Bleach), at intermediate recovery point (Half-recovery), and at the end of record fluorescent recovery (End-recovery); regions of interest (ROIs: 2 μm2 area indicated with insets). Fluorescence in ROIs was measured before bleaching and for 150 s after bleaching with argon laser at 488 nm. Cells were cultured on fibrillar Col-coated surface for 3 h. (B and C) Typical normalized FRAP curves are displayed in (B) SW480 (indigo) and SW480 KD (blue), and (C) mEF WT (gray), KO (red), and Vim rescue (magenta). Scale bar 10 μm. (D) FRAP mobile fractions, and (E) The FRAP-half-life recovery. All FRAP images were quantified with ImageJ (n = 3, at least 15 cells per group).

Article Snippet: SW480 cells, which are a cancer line that express vimentin filaments, were obtained from ATCC.

Techniques: Transfection, Fluorescence, Cell Culture

Journal: The FASEB Journal

Article Title: Vimentin‐mediated myosin 10 aggregation at tips of cell extensions drives MT1‐MMP‐dependent collagen degradation in colorectal cancer

doi: 10.1096/fj.202300672r

Figure Lengend Snippet: FIGURE 5 Vimentin colocalizes with MT1-MMP in progressive CRC. (A) Representative images of colon tissue staining with DAPI (blue), Vim (red), MT1-MMP (green), and their colocalization (magenta) areas in normal (control) tissue, low-grade, and high-grade adenocarcinoma (tumor). Images were obtained with a Zeiss Axio Scan.Z1 microscope, objective 20×. Scale bar: 100 μm. (B and C) Quantification of Pearson colocalization for Vim and MT1-MMP in normal tissue, low-grade (gray; LG), and high-grade (black; HG) tumor estimated for (B) epithelial and (C) stromal regions. (D) Representative images of MT1-MMP-Vim interaction studied by PLA (red dots) in SW480 and mEF WTs cells transfected with GFP-Myo10 plasmid (green). (E) Quantification of colocalization of PLA dots and Myo10 in SW480 and mEF cells. Paired t-test calculated for three biological replicates (presented by circle, square, and triangle symbols) with at least 15 cells per condition. (F) Representative images of Myo10-Vim interaction studied by PLA (white dots) in SW480 and mEF WTs cells, with and without Vim. (G) Quantification of positive PLA signals (Dots per cell) are shown that Vim depletion significantly reduced the number of positive PLA signals in SW480 (p < .0001) and mEF (p < .0001). Paired t-test was calculated for three biological replicates with at least 15 cells per condition. (H) MT1-MMP and Myo10 immunoprecipitations (IP) in mEF WT cells. IP of MT1-MMP was immunoblotted for Myo10. Input shows total abundance of proteins in cell lysate, and actin was used as a loading control.

Article Snippet: SW480 cells, which are a cancer line that express vimentin filaments, were obtained from ATCC.

Techniques: Staining, Control, Microscopy, Transfection, Plasmid Preparation

Journal: Computational molecular bioscience

Article Title: Analysis of Differential Gene Expression and Core Canonical Pathways Involved in the Epithelial to Mesenchymal Transition of Triple Negative Breast Cancer Cells by Ingenuity Pathway Analysis

doi: 10.4236/cmb.2023.132002

Figure Lengend Snippet: Fluorescent imaging of a non-transgenic breast cancer cell line (left) compared to the genetically engineered breast cancer cell (right). Red fluorescent demonstrates RFP labeled vimentin expression.

Article Snippet: The VIM RFP reporter cell line (ATCC HTB-26MET) was created using CRISPR/Cas9 gene editing in the parental MDA-MB-231 breast adenocarcinoma cell line (ATCC HTB-26).

Techniques: Imaging, Transgenic Assay, Labeling, Expressing

Journal: Antibiotics

Article Title: Antibacterial and Anti-Inflammatory Activity of an Antimicrobial Peptide Synthesized with D Amino Acids

doi: 10.3390/antibiotics9120840

Figure Lengend Snippet: MIC values of SET-M33D peptide on selected strains of major pathogen species.

Article Snippet: E. coli ATCC 25922 , 1.5 , 0.35 , <6.6 × 10 −10 * , 3.8 × 10 −8 ± 1.9 × 10 −8.

Techniques: Bla VIM Assay

Journal: Antibiotics

Article Title: Antibacterial and Anti-Inflammatory Activity of an Antimicrobial Peptide Synthesized with D Amino Acids

doi: 10.3390/antibiotics9120840

Figure Lengend Snippet: Frequency of selection (mean ± SD) of resistant mutants on three reference strains after exposure to SET-M33D and to colistin.

Article Snippet: E. coli ATCC 25922 , 1.5 , 0.35 , <6.6 × 10 −10 * , 3.8 × 10 −8 ± 1.9 × 10 −8.

Techniques: Selection, Mutagenesis

Journal: Antibiotics

Article Title: Antibacterial and Anti-Inflammatory Activity of an Antimicrobial Peptide Synthesized with D Amino Acids

doi: 10.3390/antibiotics9120840

Figure Lengend Snippet: Effect of SET-M33D on protein release or gene expression of proinflammatory cytokines. ( A , B ) ELISA measurement of TNF-α and IL-6 produced by RAW264.7 cells after stimulation with LPS from P. aeruginosa (20 ng/mL) or LTA from S. aureus (2 µg/mL) in the presence of different concentration of SET-M33D. Data is expressed as percentage inhibition of cytokines with respect to LPS or LTA values (100%). Values are the mean ± SD of five independent experiments (n = 5). IC50s are reported in the text. ( C – L ) Gene expression of proinflammatory cytokines MIP1, KC, IP10, TNF-α, and IL-6 was analyzed by RT-PCR. RAW264.7 cells were stimulated with LPS from E. coli ( C – G ) or with LTA from S. aureus ( H – L ) in presence of SET-M33D at 10 or 1 µM. Densitometric analysis of cDNA bands (pictures under the columns) was carried out using ImageJ software. The reduction of cDNA is indicated as fold change with respect to control ± SD of two independent experiments (n = 2). * p < 0.05, ** p < 0.01 calculated using Student’s t -test with GraphPad Prism.

Article Snippet: E. coli ATCC 25922 , 1.5 , 0.35 , <6.6 × 10 −10 * , 3.8 × 10 −8 ± 1.9 × 10 −8.

Techniques: Gene Expression, Enzyme-linked Immunosorbent Assay, Produced, Concentration Assay, Inhibition, Reverse Transcription Polymerase Chain Reaction, Software, Control

Journal: Scientific Reports

Article Title: Lack of vimentin impairs endothelial differentiation of embryonic stem cells

doi: 10.1038/srep30814

Figure Lengend Snippet: ( a ) Representative phase images are shown of WT ESCs and VIM −/− ESCs cultured on MEF feeder layers. Arrows indicate refractive edges of cell colonies. Scale bar represents 200 μm. ( b ) Gene expression of Nanog, Oct4 , and Sox2 ( all normalized to Gapdh ) are shown for both cell types. Data are presented as mean ± SEM (n = 3). ( c ) Flow cytometry analysis for both cell types of NANOG, OCT3/4, and SOX2 are shown. Shaded histograms are for staining (secondary antibody-only) controls. Values listed are for the percentage of cells within the population considered to be positive. WT ESCs are represented in black and VIM −/− ESCs are in red.

Article Snippet: Vimentin knockout mouse embryonic stem cells (VIM −/− ESCs; strain C57BL/6; Vim_AF3 from the KOMP Repository) and wild type mouse embryonic stem cells (WT ESCs; strain 129; ESD3 cells from ATCC™) were expanded as previously described .

Techniques: Cell Culture, Expressing, Flow Cytometry, Staining

Journal: Scientific Reports

Article Title: Lack of vimentin impairs endothelial differentiation of embryonic stem cells

doi: 10.1038/srep30814

Figure Lengend Snippet: ( a ) Phase images for WT EBs and VIM −/− EBs at Day 1 before (left image) and after removal (right image) from the microwells, as well as at Day 2, 4, 6 in suspension culture. All images are at the same magnification and the scale bar represents 400 μm. ( b ) Cross sectional areas for WT EBs and VIM −/− EBs were calculated from phase images (n = 50 EBs per group). ( c ) Immunohistochemical analysis indicates the proliferation marker Ki67 (green) with a nuclear counterstain (blue). Scale bar represents 200 μm.

Article Snippet: Vimentin knockout mouse embryonic stem cells (VIM −/− ESCs; strain C57BL/6; Vim_AF3 from the KOMP Repository) and wild type mouse embryonic stem cells (WT ESCs; strain 129; ESD3 cells from ATCC™) were expanded as previously described .

Techniques: Immunohistochemical staining, Marker

Journal: Scientific Reports

Article Title: Lack of vimentin impairs endothelial differentiation of embryonic stem cells

doi: 10.1038/srep30814

Figure Lengend Snippet: ( a ) Histological sections were stained for ECAD protein (green) with a nuclear counterstain (blue). The star indicates staining within the interior of a WT EB and arrows indicate discontinuous ECAD expression along VIM −/− EB outer layers. All images are at the same magnification and the scale bar represents 200 μm. ( b ) SEM images were taken of both whole and fractured WT EBs and VIM −/− EBs. Length of scale bars is indicated in each image.

Article Snippet: Vimentin knockout mouse embryonic stem cells (VIM −/− ESCs; strain C57BL/6; Vim_AF3 from the KOMP Repository) and wild type mouse embryonic stem cells (WT ESCs; strain 129; ESD3 cells from ATCC™) were expanded as previously described .

Techniques: Staining, Expressing

Journal: Scientific Reports

Article Title: Lack of vimentin impairs endothelial differentiation of embryonic stem cells

doi: 10.1038/srep30814

Figure Lengend Snippet: Gene expression of Nanog, Oct4 , and Sox2 (all normalized to Gapdh ) are shown for WT EBs and VIM −/− EBs over 7 days of differentiation as EBs. Data presented as mean ± SEM (n = 3).

Article Snippet: Vimentin knockout mouse embryonic stem cells (VIM −/− ESCs; strain C57BL/6; Vim_AF3 from the KOMP Repository) and wild type mouse embryonic stem cells (WT ESCs; strain 129; ESD3 cells from ATCC™) were expanded as previously described .

Techniques: Expressing

Journal: Scientific Reports

Article Title: Lack of vimentin impairs endothelial differentiation of embryonic stem cells

doi: 10.1038/srep30814

Figure Lengend Snippet: ( a ) Gene expression of Brachy-T (mesodermal commitment), as well as Meox1 (paraxial mesoderm) and Flk1 (lateral plate mesoderm) are shown for WT EBs and VIM −/− EBs over 7 days of differentiation (all normalized to Gapdh ). ( b ) Immunohistochemical analysis of FLK1 protein expression (green) with a nuclear counterstain (blue) in EBs at Days 6, 8, and 10. All images were taken at the same magnification and the scale bar represents 200 μm. ( c ) A representative histogram of FLK1 protein expression is shown for WT EBs (black) and VIM −/− EBs (red), as well as their respective secondary antibody-only controls (shaded histograms). The bar graph shows the percentage of positive cells for each group at Day 10. Data are presented as mean ± SEM (n = 3 for gene expression; n = 4 for protein expression) with significant differences indicated using asterisks (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: Vimentin knockout mouse embryonic stem cells (VIM −/− ESCs; strain C57BL/6; Vim_AF3 from the KOMP Repository) and wild type mouse embryonic stem cells (WT ESCs; strain 129; ESD3 cells from ATCC™) were expanded as previously described .

Techniques: Expressing, Immunohistochemical staining

Journal: Scientific Reports

Article Title: Lack of vimentin impairs endothelial differentiation of embryonic stem cells

doi: 10.1038/srep30814

Figure Lengend Snippet: ( a ) Gene expression of Tie2, Pecam , and VE-cadherin are shown for WT EBs and VIM −/− EBs over 7 days of differentiation (all normalized to Gapdh ). ( b ) Immunohistochemical analysis of TIE2, PECAM, and VE-CADHERIN protein expression (green) with a nuclear counterstain (blue) in EBs at Days 8 and 10. All images were taken at the same magnification and the scale bar represents 200 μm. ( c ) The bar graphs show the percentage of positive cells for TIE2, PECAM, and VE-CADHERIN at Day 10. Data are presented as mean ± SEM (n = 3 for gene expression; n = 4 for protein expression) with significant differences indicated using asterisks (*p<0.05, **p<0.01, ***p<0.001).

Article Snippet: Vimentin knockout mouse embryonic stem cells (VIM −/− ESCs; strain C57BL/6; Vim_AF3 from the KOMP Repository) and wild type mouse embryonic stem cells (WT ESCs; strain 129; ESD3 cells from ATCC™) were expanded as previously described .

Techniques: Expressing, Immunohistochemical staining