Journal:

Article Title: Amino-terminal Polypeptides of Vimentin Are Responsible for the Changes in Nuclear Architecture Associated with Human Immunodeficiency Virus Type 1 Protease Activity in Tissue Culture Cells

doi:

Figure Lengend Snippet: Typical optical sections of cells microinjected with the peptides and substances listed in Table ​Table1.1. All peptides were used at a concentration of 0.2 mM in 10 mM MOPS, pH 7. All panels show SW 13 [vimentin−] cells, except for C in which a SW 13 T3 M [vimentin+] cell is shown. Substances injected were MOPS buffer (A); HIV-1 PR, 10 μg/ml in 10 mM MOPS, pH 7 (B); HIV-1 PR, 10 μg/ml in 10 mM MOPS, pH 7, microinjected into an SW 13 T3 M [vimentin+] cell (C); vimentin peptide NT1 (vimentin1–96) (D); vimentin1–51 (E); vimentin1–60 (F); vimentin1–92 (G); vimentin423–465 (H); vimentin17–51 (I); vimentin17–60 (J); vimentin17–92 (K); vimentin32–51 (L); vimentin32–60 (M); vimentin32–92 (N); vimentin1-Δ(25–38)-51 (O); vimentin1-Δ(25–38)-60 (P); vimentin1-Δ(25–38)-92 (Q); vimentin1-Δ(25–63)-92 (R); vimentin1-Δ(25–68)-92 (S); vimentin1-Δ(44–68)-92 (T); peptide R23R (vimentin22–44) (U); peptide R25R (vimentin44–68) (V); peptide P410 (vimentin3–22) (W); peptide P411 (vimentin24–44) (X); and peptide P412 (vimentin83–103) (Y). Bar, 10 μm (A–Y).

Article Snippet: To get a more statistically significant overview and to delineate those residues of the vimentin peptides responsible for the effects observed, a large number of SW 13 [vimentin − ] cells were microinjected with various purified peptides, each at a final concentration of 0.2 mM, or control substances and subjected to CLS microscopy and analysis.

Techniques: Concentration Assay, Injection

Journal:

Article Title: Amino-terminal Polypeptides of Vimentin Are Responsible for the Changes in Nuclear Architecture Associated with Human Immunodeficiency Virus Type 1 Protease Activity in Tissue Culture Cells

doi:

Figure Lengend Snippet: Statistical evaluation of the effect of microinjected substances on the distribution of chromatin in the nuclei of SW 13 [vimentin − ] cells

Article Snippet: To get a more statistically significant overview and to delineate those residues of the vimentin peptides responsible for the effects observed, a large number of SW 13 [vimentin − ] cells were microinjected with various purified peptides, each at a final concentration of 0.2 mM, or control substances and subjected to CLS microscopy and analysis.

Techniques: Injection, Derivative Assay, Mutagenesis

Journal:

Article Title: Amino-terminal Polypeptides of Vimentin Are Responsible for the Changes in Nuclear Architecture Associated with Human Immunodeficiency Virus Type 1 Protease Activity in Tissue Culture Cells

doi:

Figure Lengend Snippet: Intracellular distribution of vimentin peptides and control substances after microinjection into SW 13 [vimentin−] cells. F-cys1-NT1 is found in both the cytoplasm and nucleus (A, fluorescein channel). T-vimentin (B, composite panel of both fluorescein channel [green] and propidium iodide channel [red]), visualized with an anti-vimentin antibody and an FITC-labeled second antibody, remains in the cytoplasm (fluorescein channel) and has no effect on nuclear structure or chromatin distribution (propidium iodide channel). FITC-dextrans of molecular weight 20–70,000 or higher remained in the cytoplasm and had no effect on nuclear architecture (C, composite of both fluorescein channel [green] and propidium iodide channel [red] after microinjection of MW 70,000 FITC-dextran). Smaller dextrans (i.e., MW 4000 FITC-dextran) were distributed throughout the cell, i.e., readily entered the nucleus, but had no effect on nuclear architecture (our unpublished results). Bar, 10 μm (A–C).

Article Snippet: To get a more statistically significant overview and to delineate those residues of the vimentin peptides responsible for the effects observed, a large number of SW 13 [vimentin − ] cells were microinjected with various purified peptides, each at a final concentration of 0.2 mM, or control substances and subjected to CLS microscopy and analysis.

Techniques: Control, Microinjection, Labeling, Molecular Weight

Journal:

Article Title: Amino-terminal Polypeptides of Vimentin Are Responsible for the Changes in Nuclear Architecture Associated with Human Immunodeficiency Virus Type 1 Protease Activity in Tissue Culture Cells

doi:

Figure Lengend Snippet: Presence of the cytoplasmic IF protein vimentin correlates with the occurrence of nuclear aberrations in SW 13 cells after microinjection of the HIV-1 PR. After microinjection of either SW 13 T3 M [vimentin+] cells (A and C) or SW 13 [vimentin−] cells (B and D), the cells were incubated for 30 min at 37°C and fixed; DNA was stained with propidium iodide and the preparations were examined via CLS microscopy. (A and B) Distribution of chromatin in the cells of the cell lines SW 13 T3 M [vimentin+] and SW 13 [vimentin−], respectively, after microinjection with control bacterial extract control buffer. Equatorial optical sections of the chromatin distribution of a SW 13 T3 M [vimentin+] cell (C) and of a SW 13 [vimentin−] cell (D) after microinjection with HIV-1 PR are presented. Although the effect of the action of HIV-1 PR on nuclear chromatin organization is dramatic, the effect on nuclear shape is less obvious, partially due to the more irregular shape of the nuclei of SW 13 cells in comparison to fibroblasts. Bar, 10 μm.

Article Snippet: To get a more statistically significant overview and to delineate those residues of the vimentin peptides responsible for the effects observed, a large number of SW 13 [vimentin − ] cells were microinjected with various purified peptides, each at a final concentration of 0.2 mM, or control substances and subjected to CLS microscopy and analysis.

Techniques: Microinjection, Incubation, Staining, Microscopy, Control, Comparison

Journal:

Article Title: Amino-terminal Polypeptides of Vimentin Are Responsible for the Changes in Nuclear Architecture Associated with Human Immunodeficiency Virus Type 1 Protease Activity in Tissue Culture Cells

doi:

Figure Lengend Snippet: Microinjection of the mixture of vimentin cleavage products produced in vitro by the action of HIV-1 PR or a defined vimentin peptide encompassing the entire amino-terminal head domain into SW 13 [vimentin−] cells also produces nuclear chromatin condensation and redistribution, but has no effect on nuclear shape. CLS microscopy optical sections of the chromatin distribution of a cell microinjected with HIV-1 PR (7 μg/ml) as a control (A) and a cell microinjected with a mixture of vimentin cleavage products (produced from overnight digestion of vimentin at 0.2 mg/ml) (B) demonstrate that the vimentin peptides, and not the HIV-1 PR, are directly responsible for the nuclear effects. Microinjection of the isolated amino-terminal peptide of mouse vimentin, NT 1 (vimentin residues 1–96), into SW 13 [vimentin−] cells resulted in nuclear alterations (C), similar to those seen with the vimentin cleavage product mixture or in SW 13 T3 M [vimentin+] cells injected with HIV-1 PR (Figure ​(Figure2).2). Microinjection of NT 1 into SW 13 T3 M [vimentin+] cells and incubation for 30 min at 37°C also resulted in condensation of chromatin and alteration in nuclear shape (D) and a perturbation of the vimentin IF network (our unpublished results). Injected cells were incubated for 30 min at 37°C before fixation, staining with propidium iodide and preparation for CLS microscopy. Bar, 10 μm (A–D).

Article Snippet: To get a more statistically significant overview and to delineate those residues of the vimentin peptides responsible for the effects observed, a large number of SW 13 [vimentin − ] cells were microinjected with various purified peptides, each at a final concentration of 0.2 mM, or control substances and subjected to CLS microscopy and analysis.

Techniques: Microinjection, Produced, In Vitro, Microscopy, Control, Isolation, Injection, Incubation, Staining

Journal: Nature Communications

Article Title: Vimentin filaments interact with the actin cortex in mitosis allowing normal cell division

doi: 10.1038/s41467-019-12029-4

Figure Lengend Snippet: Tailless vimentin(1-411) disrupts vimentin wt distribution and interferes with chromosomes in mitosis. a Scheme of vimentin domains with the tail sequence displayed in full. b Scheme showing the experimental strategies: bicistronic plasmids coding for DsRed2 fluorescent protein (RFP) and untagged vimentin wt (RFP//vim wt) or tailless (residues 1-411) (RFP//vim(1-411)) were transfected into vimentin-expressing cells, vim(+), or vimentin-deficient cells, vim(−), alone, for detection by immunofluorescence, or together with a small amount of the corresponding GFP-vimentin construct (GFP-vim) for direct visualization. c SW13/cl.2 human adrenocarcinoma, MCF7 breast carcinoma and HAP1 vim(−) cells transfected with the indicated constructs were observed live 48 h later. The graph shows the proportion of the cellular area occupied by every construct (* p < 10 −5 vs. wt). d SW13/cl.2 cells were transfected with the indicated amounts (in μg) of vim(1-411) (upper panels), or with different proportions of constructs coding for vimentin wt (yellow) or (1-411) (pink), as detailed in the “Methods” section (lower panels), and vimentin condensation was measured as above (* p < 10 −5 vs. 10:0 vim wt:vim(1-411); # p < 0.05 vs. 0.2 and 0.8 μg). e U-251 MG astrocytoma, Vero and SW13 parental cells were transfected with RFP//vimentin wt or RFP//vimentin(1-411). Full-length vimentin condensation was assessed by immunofluorescence with V9 anti-vimentin antibody, which recognizes the tail domain (green) (* p < 10 −7 vs. wt). f SW13/cl.2 cells were transfected with RFP//vimentin wt or (1-411) and vimentin distribution assessed by immunofluorescence. Single overlay sections are shown. g SW13/cl.2 cells were visualized live after transfection with RFP//vimentin plus GFP-vimentin wt or (1-411), as indicated. CFP-lamin A was used to delimit the nuclear envelope. Insets in f and g display overall projections of merged images. h SW13/cl.2 cells were transfected with RFP//vimentin wt or (1-411) and vimentin distribution in mitosis was observed by immunofluorescence. Single sections taken at mid-cell height (left images) and 3D-reconstructions (right images) are shown. Images in small panels below depict overall projections for vimentin alone (left) or the merge of the three channels (vimentin, RFP and DAPI). Scale bars, 20 μm. The number of determinations for the experimental conditions shown in graphs from left to right was the following: c 20, 22, 25, 23, 26, 36; d 20, 20, 20, 20, 11, 16, 13; e 20, 20, 36, 52, 30, 30. Average values ± SEM are shown. All p values were obtained with two-tailed, unpaired Student’s t -test and original datasets are provided as Source Data file

Article Snippet: SW13 parental cells (mixture of vimentin-positive and negative cells) from The European Collection of Authenticated Cell Cultures (ECACC 87031801) were acquired from Sigma.

Techniques: Sequencing, Transfection, Expressing, Immunofluorescence, Construct, Two Tailed Test

Journal: Nature Communications

Article Title: Vimentin filaments interact with the actin cortex in mitosis allowing normal cell division

doi: 10.1038/s41467-019-12029-4

Figure Lengend Snippet: Monitorization of cells expressing vimentin wt or vimentin(1-411) during mitosis. a SW13/cl.2 cells were transfected with RFP//vimentin wt plus a tracer amount of GFP-vimentin wt, or the equivalent constructs for vimentin(1-411), as indicated, for live cell monitoring by time-lapse microscopy. Several fields were randomly selected and images were acquired every 15 min. Representative images of the overlays of DIC and vimentin green fluorescence (upper panels) and green fluorescence only (lower panels, in gray scale), at the indicated time points, are shown. Scale bars, 20 μm. b Schematic representation of the main fates observed for cells transfected with each construct. c Duration of mitosis from cell rounding to separation of daughter cells (* p < 10 −6 ). d The proportion of vimentin signal present in each daughter cell is presented in different colors (* p < 10 −5 ). e SW13/cl.2 cells were transfected with RFP//vim wt or (1-411). Nuclear morphology was assessed by DAPI staining and vimentin distribution by immunofluorescence. The percentage of cells showing multiple or aberrant nuclei (arrowheads) is depicted in the lower graph (* p < 0.002). f SW13/cl.2 cells, transfected as in e , were synchronized by mild nocodazole treatment. Cells in anaphase were monitored 100 min after nocodazole removal and the proportion of cells showing lagging chromosomes, assessed by DAPI staining, is depicted in the lower graph (* p < 0.01). The number of determinations for the experimental conditions shown in graphs from left to right was the following: c 38, 25; d 24, 16; e 3 totaling 336 cells, 3 totaling 219 cells; f 4 totaling 96 cells, 4 totaling 64 cells. Average values ± SEM are shown. All p values were obtained with two-tailed, unpaired Student’s t -test and original datasets are provided as Source Data file

Article Snippet: SW13 parental cells (mixture of vimentin-positive and negative cells) from The European Collection of Authenticated Cell Cultures (ECACC 87031801) were acquired from Sigma.

Techniques: Expressing, Transfection, Construct, Time-lapse Microscopy, Fluorescence, Staining, Immunofluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: Vimentin filaments interact with the actin cortex in mitosis allowing normal cell division

doi: 10.1038/s41467-019-12029-4

Figure Lengend Snippet: Effect of microtubule or actin filament disruption on the distribution of vimentin wt and vimentin(1-411). a – d The distribution of vimentin and tubulin or f-actin was assessed in interphase and mitotic cells. a SW13/cl.2 cells transfected with RFP//vimentin wt or (1-411) to express untagged vimentin proteins, were treated in the absence or presence of 5 µM nocodazole for 30 min in serum-free medium. Vimentin (green) and tubulin (red) were visualized by immunofluorescence. b , c SW13/cl.2 cells were transfected as above, cultured in the absence b or presence c of 10 µg/ml cytochalasin B for 30 min in serum-free medium. Vimentin (green) was visualized by immunofluorescence and f-actin was stained with Phalloidin (red). Fluorescence intensity profiles of vimentin and actin along the dotted lines are shown in the right panels. d – f SW13/cl.2 cells transfected with RFP//vimentin wt were treated in serum-free medium with 2.5 µM latrunculin A d or 50 nM jasplakinolide e for 30 min, and processed by immunofluorescence. Representative images of single sections taken at mid-height of interphase and dividing cells are shown. f Quantification of the proportion of vimentin located at the cell periphery upon disruption of microtubules or actin. Noc nocodazole; CytB cytochalasin B; LatA latrunculin A; Jasp jasplakinolide (* p < 10 −7 vs. control; # p < 0.02 vs. CytB and LatA). h Vero cells were treated with the indicated agents and the distribution of endogenous vimentin and actin in mitotic cells was assessed as above. Graph shows the proportion of vimentin associated with the cell periphery (* p < 0.001 vs. control). The number of determinations for the experimental conditions shown in graphs from left to right was the following: f 14, 16, 14, 11, 28; g 25, 12, 13, 16. Average values ± SEM are shown. All p- values were obtained with two-tailed, unpaired Student’s t -test. Data are available in the Source Data file. Scale bars, 20 μm

Article Snippet: SW13 parental cells (mixture of vimentin-positive and negative cells) from The European Collection of Authenticated Cell Cultures (ECACC 87031801) were acquired from Sigma.

Techniques: Disruption, Transfection, Immunofluorescence, Cell Culture, Staining, Fluorescence, Control, Two Tailed Test

Journal: Nature Communications

Article Title: Vimentin filaments interact with the actin cortex in mitosis allowing normal cell division

doi: 10.1038/s41467-019-12029-4

Figure Lengend Snippet: Cortical association of type-III intermediate filaments in several cell types and pathophysiological conditions. a The distribution of f-actin and vimentin in interphase and mitotic cells was assessed in several tumoral or non-tumoral cell types, the characteristics of which are specified in Supplementary Table . MCF7 cells were transfected, as indicated. Representative single merged sections at mid-cell height are shown. Individual channels are depicted in Supplementary Fig. . b The distribution of GFAP and desmin was assessed by immunofluorescence in mitotic human U-251 MG astrocytoma cells and undifferentiated murine C2C12 myoblasts, respectively. Insets ( a , b ) depict enlarged areas of the cell periphery showing vimentin ( a ) or GFAP and desmin ( b ) distribution. c SW13/cl.2 cells expressing untagged vimentin wt or d Vero cells were treated with electrophilic lipids, 10 µM 4-hydroxynonenal (HNE) for 4 h or 20 µM prostaglandin A (PGA ) for 20 h, and processed as above. Graphs show the proportion of peripheral vimentin. For comparison, values corresponding to vimentin(1-411) are included in the graph in c . * p < 0.05, ** p < 10 −7 vs. control in c and * p < 10 −10 vs. control in d . The number of determinations for the experimental conditions shown in graphs from left to right was the following: c 15 for all conditions; d 26, 32, 26. Average values ± SEM are shown. All p- values were obtained with two-tailed, unpaired Student’s t -test. Source Data file contains original datasets. e SW13/cl.2 cells were transfected with a combination of RFP//vimentin wt (80%) plus a tracer amount of mCherry-vimentin wt (20%), to monitor vimentin filaments (grayscale in main images, red in insets) in live cells. Additionally, cells were transfected with the HIV-type I protease (GFP-PR, green fluorescence depicted in insets). In upper panels, cells were imaged 24 h after transfection. In the lower panels, the HIV inhibitor ritonavir was added immediately after transfection and cells were imaged 24 h later (left panel). Subsequently, ritonavir was removed and cells were imaged 5 h later (lower right panel). f Lastly, cells were fixed and stained with DAPI to identify mitotic cells. Vimentin is artificially colored in green. g Vero cells were transfected with GFP-PR (green) and 24 h later, vimentin was detected by immunofluorescence (magenta). Left images show overlay projections, right images depict vimentin distribution in gray scale. Results are representative from three experiments with similar effects. Scale bars, 20 μm

Article Snippet: SW13 parental cells (mixture of vimentin-positive and negative cells) from The European Collection of Authenticated Cell Cultures (ECACC 87031801) were acquired from Sigma.

Techniques: Transfection, Immunofluorescence, Expressing, Comparison, Control, Two Tailed Test, Fluorescence, Staining

Journal: Nature Communications

Article Title: Vimentin filaments interact with the actin cortex in mitosis allowing normal cell division

doi: 10.1038/s41467-019-12029-4

Figure Lengend Snippet: Analysis of the relative positions of vimentin and actin in mitosis by STED superresolution microscopy. a Confocal microscopy images illustrating the identification of mitotic Vero cells by the typical aspect of dividing chromosomes in DIC, confirmed by DAPI staining in merged images. The distribution of f-actin and vimentin was monitored by TRITC-phalloidin staining and immunofluorescence with Alexa488-conjugated V9 antibody, respectively. b Mitotic Vero cells, identified by DIC visualization, were analyzed by STED and images of several cells are shown. Colocalization masks (shown in white) or enlargements of areas delimited by dashed rectangles are shown to the right. Co-localization analysis was performed with Leica software. Numbers in insets represent the Pearson’s coefficient and the percentage of co-localization for the regions shown. c SW13/cl.2 cells stably transfected with RFP//vimentin wt were treated with 0.4 µM nocodazole overnight, to increase the proportion of mitotic cells. Vimentin and f-actin were detected as above. STED images of several cells are shown. Overlays with DIC images are confocal images. Colocalization masks (white) or enlargements of areas delimited by dashed rectangles are shown to the right. Numbers in images represent the Pearson’s coefficient and the percentage of co-localization, respectively, for the whole cell or for the regions enlarged. d Proportion of f-actin or vimentin located at the cell periphery ( n = 21, * p < 0.02 by two-tailed, unpaired Student’s t -test). e Colocalization of cortical vimentin with f-actin analyzed by calculation of overlap and Pearson coefficients for the cortical ROI ( n = 16). Datasets for d and e are provided in the Source Data file. Average values ± SEM are shown. f 3D-reconstruction of vimentin organization, after deconvolution of the green channel using Imaris software, for one representative cell treated as in c . g 3D-reconstruction using the basal half of the sections from the same cell in order to show the inside and the outside of the sphere. Single channels (upper panels) and merged images (lower panels) are shown. The semi-sphere edge is marked in the green channel (dotted line). The bottom-right image is a snapshot of Supplementary Movie . The arrow points at a protrusion of vimentin through the actin cortex. Scale bars, 10 μm

Article Snippet: SW13 parental cells (mixture of vimentin-positive and negative cells) from The European Collection of Authenticated Cell Cultures (ECACC 87031801) were acquired from Sigma.

Techniques: Microscopy, Confocal Microscopy, Staining, Immunofluorescence, Software, Stable Transfection, Transfection, Two Tailed Test

Journal: Nature Communications

Article Title: Vimentin filaments interact with the actin cortex in mitosis allowing normal cell division

doi: 10.1038/s41467-019-12029-4

Figure Lengend Snippet: Vimentin expression affects the properties of the actin cortex in mitosis. a Scheme of the different approaches employed to analyze mitotic cell properties. XY image stacks were used to obtain 3D-reconstructions of mitotic cells, 2D-cartographic, and orthogonal projections. b 3D-reconstruction of SW13/cl.2 cells, stably expressing vimentin wt (top), RFP from the bicistronic pIRES DsRed Express2 empty plasmid (RFP// empty plasmid, middle), or non-transfected (bottom), scale bars, 20 μm. c 2D-maps from the cells shown in (b). For the vimentin-expressing cell, the merged and single channels are shown. d Fluorescence intensity profiles along the white dotted lines drawn on the 2D-maps. e Standard deviation values of the f-actin signal of 2D-maps (* p < 0.05, *** p < 0.001). f SW13/cl.2 cells were transfected with RFP//vimentin wt (untagged vimentin wt) or RFP//vimentin(1-411) (untagged tailless vimentin), or not transfected (non-transfected). Orthogonal projections illustrate the positions of the vimentin constructs. Arrowheads mark the appearance of vimentin wt at the top and at the bottom of the cell. The inset shows an enlarged image illustrating the localization of vimentin(1-411) with respect to the cytoplasmic f-actin ring (arrow). Right panels show the top and bottom sections for every construct. Scale bars in right panels, 5 μm. g Proportion of vimentin or f-actin present at the basal layer with respect to the total cellular content (* p < 0.01 vs. cells transfected with RFP//vimentin wt). h Graph summarizing the mean and standard deviation of the f-actin signal intensity at the bottom section of the cell ( # p < 0.02 and § p < 0.01 vs. cells transfected with RFP//vimentin wt). The number of determinations for the experimental conditions shown in graphs from left to right was the following: e 25, 20, 28; g 19, 20, 20, 18, 20; h 10, 24, 13, 10, 25, 13. Average values ± SEM are shown. p- values were obtained with ANOVA followed by Tukey’s multiple comparison test in e an with the two-tailed, unpaired Student’s t -test in g and h . Data are provided in the Source Data file

Article Snippet: SW13 parental cells (mixture of vimentin-positive and negative cells) from The European Collection of Authenticated Cell Cultures (ECACC 87031801) were acquired from Sigma.

Techniques: Expressing, Stable Transfection, Plasmid Preparation, Transfection, Fluorescence, Standard Deviation, Construct, Comparison, Two Tailed Test

Journal: Nature Communications

Article Title: Vimentin filaments interact with the actin cortex in mitosis allowing normal cell division

doi: 10.1038/s41467-019-12029-4

Figure Lengend Snippet: Organization and mitotic distribution of several C-terminal truncated vimentin mutants. a Scheme of truncated vimentin mutants. b Overall projections of live SW13/cl.2 or MCF7 cells transfected with RFP//vimentin(1-423) plus GFP-vimentin(1-423). Insets, merged channels showing RFP fluorescence to delimit the cell contour. c Immunofluorescence of SW13/cl.2 cells transfected with RFP//vimentin(1-423), illustrating truncated vimentin distribution in mitosis; left image, single section at mid-cell height; right image, 3D-projection; lower panels, overall projections of vimentin alone (green) or overlays of vimentin, DAPI and RFP fluorescence. d Live cells transfected with RFP//vimentin(1-423) plus GFP-vimentin(1-423) monitored by time-lapse microscopy, illustrating asymmetric partition (upper panels) and mitotic catastrophe (lower panels). e Cells were transfected as in c and vimentin and f- actin distributions monitored in interphase (left image) and mitosis (right image; arrow, cytoplasmic f-actin); right panel, vimentin and f-actin fluorescence intensity along the dotted line in the mitotic cell. f and j SW13/cl.2 cells were transfected with RFP//vimentin(1-448) in f , or RFP//vimentin(1-459) in j , alone or alongside the corresponding GFP-fusion construct, as indicated; left panels, vimentin immunofluorescence; right panels, vimentin visualization in live cells. g and k Cells transfected with RFP//vimentin(1-448) in g , or RFP//vimentin(1-459) in k were analyzed as in c . h Time-lapse monitoring of RFP//vimentin(1-448)-transfected cells illustrating mitotic failure (upper sequence) and asymmetric division (lower sequence). l Monitorization of RFP//vimentin(1-459)-transfected cells undergoing normal (upper panel) or asymmetric division (lower panel; arrow, vimentin vestige). i and m Vimentin and f-actin distribution in resting and mitotic RFP//vimentin(1-448)- ( i ) or RFP//vimentin(1-459)-( m ) transfected cells, as described for e . n Ability of vimentin constructs to form an extended network in interphase, quantitated as percentage of cell area covered by vimentin (* p < 0.001). o Correlation between the proportion of cells with vimentin near dividing chromosomes ( x -axis) for each vimentin-truncated mutant and the corresponding mitosis duration (left y -axis, red labels, y = 4.7251 x − 105.33; R 2 = 0.899) or the proportion of cells showing multiple/aberrant nuclei (right y -axis, blue labels, y = 4.4633 x − 47.031; R 2 = 0.994) (* p < 0.01; # p < 0.02). The number of determinations for the experimental conditions shown in graphs from left to right was the following: n 22, 26, 36, 39, 20 o duration of mitosis, 17, 15, 30; vimentin near chromosomes, three determinations totaling 51, 55, and 51 cells; cells with multiple or aberrant nuclei, three determinations totaling 155, 206, and 151 cells. Average values ± SEM are shown. All p values were obtained by two-tailed, unpaired Student’s t -test. Scale bars, 20 μm. Data are available in the Source Data file

Article Snippet: SW13 parental cells (mixture of vimentin-positive and negative cells) from The European Collection of Authenticated Cell Cultures (ECACC 87031801) were acquired from Sigma.

Techniques: Transfection, Fluorescence, Immunofluorescence, Time-lapse Microscopy, Construct, Sequencing, Mutagenesis, Two Tailed Test

Journal: Nature Communications

Article Title: Vimentin filaments interact with the actin cortex in mitosis allowing normal cell division

doi: 10.1038/s41467-019-12029-4

Figure Lengend Snippet: Assembly and distribution of GFP fusion constructs of truncated vimentin forms. a Live confocal microscopy assessment of the morphology of vimentin assemblies 48 h after transfection of SW13/cl.2 cells with the constructs schematized in panel b . Overall projections are shown. Insets show enlarged areas of interest. The graph (right) depicts the percentage of cells with squiggles or short filaments for every construct (* p < 10 −6 vs. wt) by two-tailed, unpaired Student’s t -test. c Cells were transfected with the indicated constructs, treated overnight with 0.4 µM nocodazole in complete medium to increase the proportion of mitotic cells, fixed, and the distribution of vimentin and f-actin in mitotic cells, assessed. Nuclei were counterstained with DAPI. Single sections taken at mid-cell height are shown in all cases. For every condition, fluorescence intensity profiles for vimentin and f-actin along the dotted lines are shown on the right. d Proportion of vimentin dots associated with the cell periphery for every construct. In this case, GFP-vimentin C328S, which contains an intact tail domain but assembles in dots, is used as a control. GFP-vimentin(1-459) is excluded from this graph due to its organization mainly in squiggles or short filaments (* p < 0.001 vs. GFP-vimentin C328S and GFP-vimentin G452V; # p < 0.001 vs. GFP-vimentin(1-448) by ANOVA followed by Tukey’s post-test for multiple comparisons). e Proportion of peripheral vimentin for all constructs (* p < 0.001 vs. GFP-vimentin wt; § p < 0.01; # p < 0.01 vs GFP-vimentin(1-448) and (1-459); † p < 0.05 by ANOVA followed by Tukey’s post-test). The number of determinations for the experimental conditions shown in graphs from left to right was the following: a 15, 15, 18, 20 and 28 fields, totaling 162, 55, 77, 97 and 142 cells, respectively; d 12, 10, 10, 15, 19; e 15, 10, 10, 12, 12, 13, 20. Average values ± SEM are shown. Data are provided in the Source Data file. Scale bars, 20 μm

Article Snippet: SW13 parental cells (mixture of vimentin-positive and negative cells) from The European Collection of Authenticated Cell Cultures (ECACC 87031801) were acquired from Sigma.

Techniques: Construct, Confocal Microscopy, Transfection, Two Tailed Test, Fluorescence, Control

Journal: PLoS ONE

Article Title: ZNF367 Inhibits Cancer Progression and Is Targeted by miR-195

doi: 10.1371/journal.pone.0101423

Figure Lengend Snippet: (A) ZNF367 knockdown increases cellular proliferation. The Y axis represents relative fluorescent units (RFU), and the X axis indicates days post-transfection. *p<0.05 relative to the negative control. Error bars represent ± SD. (B) ZNF367 knockdown enhances tumor growth in vivo. SW13 cells were transfected with the negative control (n = 4) and siRNA (n = 4) into the right and left flank of each mouse. After 48 hours of transfection, 3×10 6 cells were injected in athymic nude mice, and tumor growth was measured weekly. The Y axis represents the tumor volume and X axis the weeks of tumor measurement after flank injection. *p<0.05 and error bars represent ± SD.

Article Snippet: Wild-type ZNF367 3′UTR or mutant ZNF367 3′UTR and the empty 3′UTR vector with pre-miR-195 or pre-NC were co-transfected into SW13 cells (Switchgear Genomics).

Techniques: Knockdown, Transfection, Negative Control, In Vivo, Injection

Journal: PLoS ONE

Article Title: ZNF367 Inhibits Cancer Progression and Is Targeted by miR-195

doi: 10.1371/journal.pone.0101423

Figure Lengend Snippet: ZNF367 overexpression decreases cellular invasion and migration. ( A ) SW13, ( B ) BD104A, ( C ) TPC-1, and ( D ) HEK293 cell lines. After transfection, cells were plated inside a Boyden chamber for 48 hours. Cells were stained and counted in 4 fields. The left panel shows the representative image (12.5X) from the siRNA knockdown and negative control groups. The right panel indicates the quantitative measurement of invaded and migrated cells in knockdown and the negative control. *p<0.05 and error bars indicate ± SD. ZNF367 overexpression decreases colony number, cellular invasion, and migration in HEK293 cells. ( E ) Western blot of ZNF367 overexpression in HEK293 and SW13 cells (Empty vector, XL4). GAPDH was used as a loading control. ( F ) Representative clonogenic image for cells with ectopic ZNF367 expression and its corresponding control (Empty vector, XL4). ( G ) Cellular invasion and migration decreased with ZNF367 overexpression in HEK293 cells. The quantitative measurement of invaded and migrated cells per group (HEK293-HEK293-Empty vector or ZNF367 ) is represented in the bar graph on the right panel. ( H ) The extracellular protein attachment of SW13 cells with ZNF367 knockdown. Cells were transfected with ZNF367 siRNA and negative control siRNA, and were plated into adhesion plates and incubated for 90 minutes. The Y axis represents absorbance at 540 nM of cells adherent to each protein. Error bars represent ± SEM. *p<0.05, ***p<0.001.

Article Snippet: Wild-type ZNF367 3′UTR or mutant ZNF367 3′UTR and the empty 3′UTR vector with pre-miR-195 or pre-NC were co-transfected into SW13 cells (Switchgear Genomics).

Techniques: Over Expression, Migration, Transfection, Staining, Knockdown, Negative Control, Western Blot, Plasmid Preparation, Control, Expressing, Incubation

Journal: PLoS ONE

Article Title: ZNF367 Inhibits Cancer Progression and Is Targeted by miR-195

doi: 10.1371/journal.pone.0101423

Figure Lengend Snippet: ( A ) ZNF367 knockdown upregulates ITGA3 expression in SW13 cells. Error bars represent ± SEM. ( B ) The correlation between ITGA3 and ZNF367 mRNA expression in adrenocortical tumor samples. X and Y axes represent log 2–transformed values. ( C ) Western blot quantification of ITGA3 protein expression with ZNF367 knockdown. ( D – E ) ITGA3 expression with ZNF367 overexpression. Error bars represent ± SEM.

Article Snippet: Wild-type ZNF367 3′UTR or mutant ZNF367 3′UTR and the empty 3′UTR vector with pre-miR-195 or pre-NC were co-transfected into SW13 cells (Switchgear Genomics).

Techniques: Knockdown, Expressing, Transformation Assay, Western Blot, Over Expression

Journal: PLoS ONE

Article Title: ZNF367 Inhibits Cancer Progression and Is Targeted by miR-195

doi: 10.1371/journal.pone.0101423

Figure Lengend Snippet: ( A ) The correlation between miR-195 and ZNF367 expression in an adrenocortical tumor. The Pearson correlation coefficient is indicated by r with its p value. ( B ) Ectopic overexpression of pre-miR-195 results in downregulation of ZNF367 . ZNF367 mRNA expression in SW13 cells transfected with pre-miR-195 and the pre-negative control at 5 nM for 24 hours (presented as a fold-change relative to the pre-negative control). Error bars represent ± SEM. The right panel shows the Western blot of the ZNF367 protein from SW13 cells transfected with pre-miR-195 and the negative control. ( C ) Pre-miR-195 overexpression increases invasion in SW13 cells compared to the negative control. The right panel shows the mean number of invaded cells on the Y axis. ( D ) ITGA3 mRNA expression is increased after transfection of miR-195 and the negative control in SW13 cells. Error bars represent ± SEM. ( E ) The binding site of miR-195 in the ZNF367 3′UTR, along with the mutant construct in the predicted seed region. In the right panel, the luciferase assay demonstrates decreased luminescence in SW13 cells co-transfected with miR-195 and the negative control at 5 nM, with the empty vector, wild-type ZNF367 3′UTR, or MUT- ZNF367 3′UTR vector (mutated in the first three nucleotides of the seed sequence). The luminescence was read after 24 hours of transfection. The Y axis represents the ratio of ZNF367 3′UTR to the empty vector. *p value < 0.05 compared to the negative control. Error bars represent ± SEM.

Article Snippet: Wild-type ZNF367 3′UTR or mutant ZNF367 3′UTR and the empty 3′UTR vector with pre-miR-195 or pre-NC were co-transfected into SW13 cells (Switchgear Genomics).

Techniques: Expressing, Over Expression, Transfection, Negative Control, Western Blot, Binding Assay, Mutagenesis, Construct, Luciferase, Plasmid Preparation, Sequencing

Journal: Cell reports

Article Title: Crimean-Congo Hemorrhagic Fever Virus suppresses Innate Immune Responses via a Ubiquitin and ISG15 Specific Protease

doi: 10.1016/j.celrep.2017.08.040

Figure Lengend Snippet: Wild-type and mutant CCHFV growth kinetics were measured by TCID50. (A) A549, BSR-T7/5, Huh7, SW13, and HAP1 cells were infected with CCHFV at an MOI of 0.01. Monocyte-derived macrophages were infected with CCHFV (MOI 1). Data are mean ± SD of two independent experiments. * represent p < 0.05 between WT and OTU mutants. (B) HUVEC and primary skin fibroblast cells were infected at an MOI of 0.5. Data are mean ± SD of three biological replicates.

Article Snippet: Conzelmann (Ludwig-Maximilians-Universität, Munich, Germany), SW13 cells from P. Leyssen (Rega Instituut KU Leuven, Belgium).

Techniques: Mutagenesis, Infection, Derivative Assay

Journal:

Article Title: DNA Damage Modulates Nucleolar Interaction of the Werner Protein with the AAA ATPase p97/VCP

doi: 10.1091/mbc.E03-02-0111

Figure Lengend Snippet: VCP and WRNp reciprocally coimmunoprecipitate. Nuclear extracts were immunoprecipitated and immunoblotted as described in MATERIALS AND METHODS. Equal amounts of total protein were immunoprecipitated with 20 μl of rabbit anti-Werner helicase (RbαW) and either the chicken (1469) or rabbit (5860) anti-VCP polyclonal antibodies. Rabbit IgG was used as a negative control. Immunoprecipitated proteins (40 μl/lane) were electrophoresed on a 7.5% polyacrylamide gel and then immunoblotted to polyvinylidene difluoride membranes. After a 1 h incubation with primary antibodies and appropriate horseradish peroxidase-conjugated secondary antibodies, proteins were visualized by enhanced chemiluminescence. The results are presented as a composite image of Jurkat (lanes 7–10), MO59K (lanes 11 and 12) or K562 (lane 13) precipitates, or total cell lysates of MO56K (lane 5) or SW13 (lane 6) cells. Lanes 1–4 contain purified bovine liver VCP, 0.5 μg, immunoblotted with chicken anti-VCP 1469 (1:2000, lane 1), or anti-VCP mAb (lane 4). As a control, 2.0 μg of purified VCP was immunoblotted with 10 μg/ml preimmune chicken serum (preimmune 1:200, lane 2) or 10 μg/ml purified chicken IgY (IgY, lane 3). The expected position of VCP and WRNp are indicated on the right and molecular masses in kilodaltons are in the middle. In some cell lysates (example, lane 5), an ∼60-kDa protein, apparently a VCP fragment, is detected by anti-VCP antibody. VCP multimers are occasionally detected, for example, in purified VCP (lane 1).

Article Snippet: Rabbit anti-WRN1 was purchased from Novus (Littleton, CO) and mouse anti-WRNp mAb and SW13 cell lysate were from BD Transduction Laboratories (San Diego, CA).

Techniques: Immunoprecipitation, Negative Control, Incubation, Purification

Journal:

Article Title: DNA Damage Modulates Nucleolar Interaction of the Werner Protein with the AAA ATPase p97/VCP

doi: 10.1091/mbc.E03-02-0111

Figure Lengend Snippet: CPT treatment effects VCP and WRNp coimmunoprecipitates. MO59K cells were treated with 10 μM CPT for 1 or 4 h or were untreated controls (0 h). In all panels, the molecular mass in kilodaltons is indicated to the right or left. (A) Rabbit anti-WRNp and rabbit anti-VCP precipitates were treated as described in Figure 4 and Western blotted with mouse anti-WRNp (lanes 1–4) or chicken anti-VCP (lanes 5 and 6) as described. (B) A single immunoblot from the same experiment of VCP (lane 3) and WRNp (lanes 4–6) IPs probed with chicken anti-VCP. Lane 1, 0.5 μg of purified bovine liver VCP; lane 2, MO59K total cell lysate (40 μg). (C) Mouse anti-WRNp detects WRNp in rabbit anti-WRNp precipitates (lanes 1–3) and in total nuclear extract (lanes 7–9), but not in rabbit anti-BRCA1 IPs (lanes 4–6). Cells were treated with CPT and Western blotted as described in text. (D) Cells were treated as described above with CPT and extracts were immunoblotted with chicken 1469 anti-VCP as described in text. Three different experiments are shown, one performed with K562 extracts (lanes 1–6) and two with MO59K extracts (lanes 8–13 and 15–20). Lanes 7 and 14, 0.5 μg of purified bovine liver VCP. (E) WRNp was detected in rabbit anti-Ku 80 precipitates (lanes 6–8) and Ku-80 was detected in rabbit anti-WRNp precipitates (lanes 2–4) of nuclear extracts. Lane 1, 20 μl of SW13 total lysate; lane 5, 20 μl of MO59K total lysate. (F) BRCA1 detected in VCP, but not WRNp IPs. MO59K cells were separated into cytoplasmic and nuclear extracts as described in MATERIALS AND METHODS and precipitated with rabbit anti-WRNp or Rb 5860 anti-VCP and probed for BRCA1.

Article Snippet: Rabbit anti-WRN1 was purchased from Novus (Littleton, CO) and mouse anti-WRNp mAb and SW13 cell lysate were from BD Transduction Laboratories (San Diego, CA).

Techniques: Western Blot, Purification