lifeact egfp plasmid construct  (Amaxa)

Journal: European Journal of Immunology

Article Title: CD3xHER2 bsAb‐Mediated Activation of Resting T‐cells at HER2 Positive Tumor Clusters Is Sufficient to Trigger Bystander Eradication of Distant HER2 Negative Clusters Through IFNγ and TNFα

doi: 10.1002/eji.202451589

Figure Lengend Snippet: Development of HER2 mosaic ECM‐embedded 3D tumoroids. (A) IHC showing different levels of HER2 expression (brown) in human breast cancer sections. Black triangle = HER2 + cancer cells. White triangle = HER2 − cancer cells. The HER2 score was determined by pathologists at Erasmus Medical Center according to HercepTest guidelines. Scale bar = 200 µm. (B) 3D confocal imaging of ECM embedded HER2 mosaic tumoroids at day 0 and day 5 after BT474 cell cluster injection. HER2 + cells (BT474 WT, Lifeact‐EGFP‐transduced, green) and HER2 − cells (BT474 HER2 KO, Hoechst33342‐labeled, blue) were mixed at the indicated ratios and 100% HER2 + and 100% HER2 − tumoroids were used as controls. The images show maximum projections. Scale bar = 100 µm. (C, D) Quantification of confocal imaging data shown in (B). (C) Fractions of HER2 + and HER2 − cells in (mosaic) tumoroids on day 0 and day 5. (D) Fold change in numbers of HER2 + and HER2 − cells in (mosaic) tumoroids at day 5 calculated relative to day 0. Values above 1 indicate cell proliferation. The graph represents three independent experiments, each performed with one co‐culture per condition. Mean ± SEM is shown. Two‐way ANOVA followed by Tukey's multiple comparisons test was performed. ns = nonsignificant.

Article Snippet: BT474 cells were transduced with a pLenti‐Lifeact EGFP construct (#187686, Addgene) and sorted using a cell sorter (LE‐SH800SFP, SONY).

Techniques: Expressing, Imaging, Injection, Labeling, Co-Culture Assay

Journal: European Journal of Immunology

Article Title: CD3xHER2 bsAb‐Mediated Activation of Resting T‐cells at HER2 Positive Tumor Clusters Is Sufficient to Trigger Bystander Eradication of Distant HER2 Negative Clusters Through IFNγ and TNFα

doi: 10.1002/eji.202451589

Figure Lengend Snippet: Proportion of HER2 − cells in mosaic tumoroids compatible with bystander killing. (A) Schematic representation of the experimental setup to investigate bystander killing. (B) Maximum projections of 3D confocal imaging of 100% HER2 + , 100% HER2 − , and HER2 mosaic tumoroids with the indicated HER2 + :HER2 − ratios at day 0, 2, and 5 after exposure to T‐cell/CD3xHER2 bsAbs mixture. The white dotted circle indicates tumoroid area. Green = BT474 WT, Lifeact‐EGFP‐transduced; blue = BT474 HER2 KO, Hoechst33342‐labeled; yellow = T‐cells, CellTracker Deep Red‐labeled; red = dead cells, PI stained. Scale bar = 100 µm. (C) Absolute T‐cell number recruited to the tumoroids on day 2 after exposure to T‐cell/CD3xHER2 bsAb mixture as shown in (B). The graph represents three independent experiments, each performed with 1–2 co‐cultures per condition, with each co‐culture generated in an individual well. Mean ± SEM is shown. (D) Fold change in number of HER2 + and HER2 − cells in tumoroids at day 5 after exposure to T‐cell/CD3xHER2 bsAb mixture calculated relative to day 0. Values below 1 indicate loss of cells. The graph represents three independent experiments, each performed with two co‐cultures per condition, with each co‐culture generated in an individual well. Mean ± SEM is shown. Two‐way ANOVA followed by Dunnett's multiple comparisons test was performed. * p < 0.05; *** p < 0.001.

Article Snippet: BT474 cells were transduced with a pLenti‐Lifeact EGFP construct (#187686, Addgene) and sorted using a cell sorter (LE‐SH800SFP, SONY).

Techniques: Imaging, Labeling, Staining, Co-Culture Assay, Generated

Journal: European Journal of Immunology

Article Title: CD3xHER2 bsAb‐Mediated Activation of Resting T‐cells at HER2 Positive Tumor Clusters Is Sufficient to Trigger Bystander Eradication of Distant HER2 Negative Clusters Through IFNγ and TNFα

doi: 10.1002/eji.202451589

Figure Lengend Snippet: Determining the reach of bystander killing from HER2 + to HER2 − islands. (A) Representative images of HER2 IHC illustrating spatial separation of HER2 + (brown) and HER2 − islands in human breast cancer sections. Distances (center‐to‐center) between HER2 + and HER2 − islands (black squares) are indicated. Scale bar = 200 µm (left panel) or 100 µm (right panel) and smaller panels are zoomed in on boxed areas. (B) Left, a cartoon showing a schematic representation of the experimental setup to investigate the reach of bystander killing; right, representative 3D confocal images (maximum projections) showing co‐printed HER2 + (green) and HER2 − (blue) tumoroids spaced at distances between 200 µm and 300 µm (center‐to‐center) in collagen matrices. (C) 3D confocal imaging (maximum projections) of HER2 + and HER2 − tumoroids printed at the indicated distances before (day 0) and 5 days after exposure to T‐cell/CD3xHER2 bsAbs mixture compared with exposure to T‐cells alone. Green = BT474 WT, Lifeact‐EGFP‐transduced; blue = BT474 HER2 KO, Hoechst33342‐labeled; red = dead cells, PI stained. Scale bar = 100 µm. (D) Fold change in HER2 + and HER2 − cell numbers at day 5 after exposure to T‐cell/CD3xHER2 bsAb mixture calculated relative to day 0. Values below 1 indicate loss of cells. The graph represents three (T cells only) or four (T‐cell/CD3xHER2 bsAb mixture) independent experiments, each performed with one co‐culture. Mean ± SEM is shown. Two‐way ANOVA followed by Dunnett's multiple comparisons test was performed. ns = non‐significant; * p < 0.05; ** p < 0.01.

Article Snippet: BT474 cells were transduced with a pLenti‐Lifeact EGFP construct (#187686, Addgene) and sorted using a cell sorter (LE‐SH800SFP, SONY).

Techniques: Imaging, Labeling, Staining, Co-Culture Assay

Journal: European Journal of Immunology

Article Title: CD3xHER2 bsAb‐Mediated Activation of Resting T‐cells at HER2 Positive Tumor Clusters Is Sufficient to Trigger Bystander Eradication of Distant HER2 Negative Clusters Through IFNγ and TNFα

doi: 10.1002/eji.202451589

Figure Lengend Snippet: Experimental design to investigate spatial and temporal aspects of bystander killing. (A) Schematic representation of the experimental design (model 1–4) to investigate spatial and temporal aspects of bystander killing. (B) 3D confocal imaging (maximum projections) showing HER2 + tumoroids (green), HER2 − tumoroids (blue), and T‐cell clusters (yellow) co‐printed at specific positions in collagen matrices in the different models as indicated in (A). (C) 3D confocal imaging (maximum projections) showing tumor killing (left) and T‐cell recruitment (right) upon exposure to CD3xHER2 bsAbs for Model 1 and 2 followed over a time course of 40 h (note the T‐cell clusters are below the imaged area). Green = BT474 WT, Lifeact‐EGFP‐transduced; blue = BT474 HER2 KO, Hoechst33342‐labeled; yellow = T‐cells, CellTracker Deep Red‐labeled; red = dead cells, PI stained. Scale bar = 100 µm. (D) Quantification of T‐cell mediated tumor killing upon exposure to CD3xHER2 bsAbs for Models 1 and 2 followed over 40 h. Graphics show fold change in HER2 + (left) and HER2 − cell numbers (right) relative to 0 h. The light green bar indicates a 50% reduction in HER2 + cell number. The graph represents two independent experiments, each performed with 1–2 co‐cultures, with each co‐culture generated in an individual well. Mean ± SEM is shown. (E) Quantification of T‐cell recruitment upon exposure to CD3xHER2 bsAbs for Models 1 and 2 followed over 40 h. Graphs show absolute T‐cell numbers within HER2 + (left) and HER2 − tumoroids (right). The graph represents two independent experiments, each performed with 1–2 co‐cultures, with each co‐culture generated in an individual well. Mean ± SEM is shown.

Article Snippet: BT474 cells were transduced with a pLenti‐Lifeact EGFP construct (#187686, Addgene) and sorted using a cell sorter (LE‐SH800SFP, SONY).

Techniques: Imaging, Labeling, Staining, Co-Culture Assay, Generated

Journal: European Journal of Immunology

Article Title: CD3xHER2 bsAb‐Mediated Activation of Resting T‐cells at HER2 Positive Tumor Clusters Is Sufficient to Trigger Bystander Eradication of Distant HER2 Negative Clusters Through IFNγ and TNFα

doi: 10.1002/eji.202451589

Figure Lengend Snippet: Spatiotemporal aspects of bystander killing after bsAb‐induced, T‐cell mediated antitumor cytotoxicity. (A, B) 3D confocal images (maximum projections) showing tumor killing (upper panel) and T‐cell recruitment (lower panel) upon exposure to CD3xHER2 bsAbs for Model 3 (A) and 4 (B) monitored over a time course of 40 h (note the T‐cell clusters are below the imaged area). Green = BT474 WT, Lifeact‐EGFP‐transduced; blue = BT474 HER2 KO, Hoechst33342‐labeled; yellow = T‐cells, CellTracker Deep Red‐labeled; red = dead cells, PI staining. Scale bar = 100 µm. (C) Quantification of T‐cell mediated tumor killing upon exposure to CD3xHER2 bsAbs for Model 3 (left) and 4 (right) followed over 40 h. Graphs show fold change in HER2 + and HER2 − cell numbers relative to 0 h. Light green bars indicate a 50% reduction in HER2 + cell number and light blue bars indicate a 50% reduction in HER2 − cell number. The graph represents two independent experiments, each performed with 1–2 (Model 3) or 2 (Model 4) co‐cultures, with each co‐culture generated in an individual well. Mean ± SEM is shown. (D) Quantification of T‐cell recruitment in HER2 + and HER2 − tumoroids upon exposure to CD3xHER2 bsAbs for Model 3 (left) and 4 (right) followed over 40 h. Graphs show absolute T‐cell numbers within HER2 + and HER2 − tumoroids. The graph represents two independent experiments, each performed with 1–2 (Model 3) or 2 (Model 4) co‐cultures, with each co‐culture generated in an individual well. Mean ± SEM is shown.

Article Snippet: BT474 cells were transduced with a pLenti‐Lifeact EGFP construct (#187686, Addgene) and sorted using a cell sorter (LE‐SH800SFP, SONY).

Techniques: Labeling, Staining, Co-Culture Assay, Generated

Journal: European Journal of Immunology

Article Title: CD3xHER2 bsAb‐Mediated Activation of Resting T‐cells at HER2 Positive Tumor Clusters Is Sufficient to Trigger Bystander Eradication of Distant HER2 Negative Clusters Through IFNγ and TNFα

doi: 10.1002/eji.202451589

Figure Lengend Snippet: Neutralizing antibodies against IFNγ and TNFα block bystander killing of HER2‐ tumoroids after T‐cell‐mediated killing of HER2+ tumoroids via bsAbs. (A) Schematic representation of the experimental design based on Model 3 (see Figure ), with or without neutralizing antibodies. (B) Maximum projections of 3D confocal imaging showing HER2 + and HER2 − tumoroids co‐printed as described in (A) and treated with or without the indicated concentrations of an IFNγ neutralizing antibody. Images were taken 6, 18, and 30 h after exposure to CD3xHER2 bsAbs. Green = BT474 WT, Lifeact‐EGFP‐transduced; blue = BT474 HER2 KO, Hoechst33342‐labeled; yellow = T‐cells, CellTracker Deep Red‐labeled; red = dead cells, PI stained. Scale bar = 100 µm. (C) Quantification of T‐cell mediated tumor killing upon exposure to CD3xHER2 bsAbs in the presence or absence of IFNγ neutralizing antibody as shown in (B). Graphs show fold change in HER2 + and HER2 − cell numbers at the indicated time points relative to 6 h. The graph represents three independent experiments, each performed with 2 co‐cultures, with each co‐culture generated in an individual well. Mean ± SEM is shown. Two‐way ANOVA followed by Dunnett's multiple comparisons test was performed. *** p < 0.001. ns = non‐significant. (D) Maximum projections of 3D confocal imaging of HER2 + and HER2 − tumoroids co‐printed as described in (A) and treated with or without 10 µg/mL TNFα neutralizing antibody, or a combination of 10 µg/mL IFNγ and 10 µg/mL TNFα neutralizing antibodies. A co‐culture without bsAb treatment serves as a negative control. Images were taken over a 50 h time course ( note : T‐cell signals were excluded from these images). Green = BT474 WT, Lifeact‐EGFP‐transduced; blue = BT474 HER2 KO, Hoechst33342‐labeled; red = dead cells, PI stained. Scale bar = 100 µm. (E) Quantification of T‐cell‐mediated tumor killing over time as shown in (D). Graphs show fold change in HER2 + and HER2 − cell numbers at the indicated time points relative to 0 h. The graph represents two independent experiments, each performed with 1–2 co‐cultures, with each co‐culture generated in an individual well. Mean ± SEM is shown. Two‐way ANOVA followed by Dunnett's multiple comparisons test was performed. * p < 0.05; ** p < 0.01; *** p < 0.001 compared with CTR. ns = non‐significant.

Article Snippet: BT474 cells were transduced with a pLenti‐Lifeact EGFP construct (#187686, Addgene) and sorted using a cell sorter (LE‐SH800SFP, SONY).

Techniques: Blocking Assay, Imaging, Labeling, Staining, Co-Culture Assay, Generated, Negative Control

Journal: bioRxiv

Article Title: Human cytomegalovirus nuclear capsid motility is non-directed and independent of nuclear actin bundles

doi: 10.1101/641266

Figure Lengend Snippet: BJ-SFFV-LifeAct-mCherry-NLS cells were infected with HCMV-TB40/e-UL32EGFP-UL100mCherry, fixed at 24hpi and stained for DNA (Hoechst) and HCMV-IE (Anti-IE1). (A) Representative image of very rare nuclear actin filaments. Scale bar indicates 10 µm. (B) Quantification of the filament rates. Large tiles spanning 0.75×0.75 µm were acquired and quantified in ImageJ and Python using scripts (see Materials and Methods). Filaments were manually counted. Means of three independent replicates are shown. Bars indicate standard deviations.

Article Snippet: Since this UL77-mGFP fusion already occupied the GFP channel, we exchanged GFP in the original LifeAct construct (RRID: Addgene_58467) with the red-fluorescent mCherry, resulting in cells showing a homogenous nuclear LifeAct signal which was slightly enriched in what seemed to be nucleoli.

Techniques: Infection, Staining

Journal: bioRxiv

Article Title: Human cytomegalovirus nuclear capsid motility is non-directed and independent of nuclear actin bundles

doi: 10.1101/641266

Figure Lengend Snippet: BJ-SFFV-Life-Act-mCherry-NLS cells were infected with HCMV-TB40/e-UL32EGFP-UL100mCherry, fixed at the indicated time points and stained for DNA (Hoechst) and HCMV-IE (Anti-IE1). pUL32-EGFP serves as a marker for late gene expression. (A) LifeAct-mCherry-NLS signal intensity drops with ongoing infection. Scale bars indicate 10 µm. (B) Quantification of subcellular Life-Act vs. IE signal intensities at 24, 48 and 72hpi using automated microscopy image analysis. (C) LifeAct intensity over time. For comparison, all image intensities are scaled to the same level in and . Scale bars indicate 10 µm.

Article Snippet: Since this UL77-mGFP fusion already occupied the GFP channel, we exchanged GFP in the original LifeAct construct (RRID: Addgene_58467) with the red-fluorescent mCherry, resulting in cells showing a homogenous nuclear LifeAct signal which was slightly enriched in what seemed to be nucleoli.

Techniques: Infection, Staining, Marker, Expressing, Microscopy

Journal: bioRxiv

Article Title: Human cytomegalovirus nuclear capsid motility is non-directed and independent of nuclear actin bundles

doi: 10.1101/641266

Figure Lengend Snippet: BJ-CMV-LifeAct-mCherry-NLS cells were infected with HCMV-TB40/e-UL32EGFP-UL100mCherry, fixed at the indicated time points and stained for DNA (Hoechst) and HCMV-IE (Anti-IE1). pUL32-EGFP serves as a marker for late gene expression. (A) LifeAct-mCherry-NLS signal intensity drops with ongoing infection. Scale bars indicate 10 µm. (B) Quantification of subcellular LifeAct vs. IE signal intensities at 24, 48 and 72hpi by automated microscopy image analysis. (C) LifeAct intensity over time. All image intensities are scaled to the same level in and for comparison. Scale bars indicate 10 µm.

Article Snippet: Since this UL77-mGFP fusion already occupied the GFP channel, we exchanged GFP in the original LifeAct construct (RRID: Addgene_58467) with the red-fluorescent mCherry, resulting in cells showing a homogenous nuclear LifeAct signal which was slightly enriched in what seemed to be nucleoli.

Techniques: Infection, Staining, Marker, Expressing, Microscopy

Journal: bioRxiv

Article Title: Human cytomegalovirus nuclear capsid motility is non-directed and independent of nuclear actin bundles

doi: 10.1101/641266

Figure Lengend Snippet: BJ-CMV-LifeAct-mCherry-NLS cells were infected with HCMV-TB40/e-UL32EGFP-UL100mCherry, fixed at the indicated time points and stained for DNA (Hoechst) and HCMV-IE (Anti-IE1). (A-D) Rate of LifeAct nuclear filaments in infected IE-1-positive cells. Four replicates with different base-line expression levels of LifeAct are shown as quantified in (E). (F) Difference in LifeAct-mCherry-NLS signal intensity in the cells with filaments (+), compared to those without (-).

Article Snippet: Since this UL77-mGFP fusion already occupied the GFP channel, we exchanged GFP in the original LifeAct construct (RRID: Addgene_58467) with the red-fluorescent mCherry, resulting in cells showing a homogenous nuclear LifeAct signal which was slightly enriched in what seemed to be nucleoli.

Techniques: Infection, Staining, Expressing

Journal: bioRxiv

Article Title: Human cytomegalovirus nuclear capsid motility is non-directed and independent of nuclear actin bundles

doi: 10.1101/641266

Figure Lengend Snippet: BJ-CMV-LifeAct-mCherry-NLS cells were infected with HCMV-TB40/e-UL32EGFP-UL100mCherry, fixed at 72hpi and stained for DNA (Hoechst) and HCMV-IE (Anti-IE1). pUL32-EGFP serves as a marker for late gene expression. (A ) Cells that show pUL32-EGFP expression have very little LifeAct-mCherry-NLS signal. Scale bar indicates 20 µm. (B) Scatter plot of nuclear LifeAct-mCherry-NLS vs. pUL32-EGFP signal compared to the nuclear mCherry signal.

Article Snippet: Since this UL77-mGFP fusion already occupied the GFP channel, we exchanged GFP in the original LifeAct construct (RRID: Addgene_58467) with the red-fluorescent mCherry, resulting in cells showing a homogenous nuclear LifeAct signal which was slightly enriched in what seemed to be nucleoli.

Techniques: Infection, Staining, Marker, Expressing

Journal: bioRxiv

Article Title: Human cytomegalovirus nuclear capsid motility is non-directed and independent of nuclear actin bundles

doi: 10.1101/641266

Figure Lengend Snippet: WT BJ and BJ-CMV-LifeAct-mCherry-NLS were infected with HCMV-HB5-UL77-mGFP at an MOI of 10 for 24 hours, fixed, and stained for IE1, as well as with Alexa-488-Phalloidin. The arrow indicates a representative BJ-CMV-LifeAct-mCherry-NLS cell in which the same nuclear actin structures are stained by both LifeAct as well as by Phalloidin. Scale bar is 10 µm.

Article Snippet: Since this UL77-mGFP fusion already occupied the GFP channel, we exchanged GFP in the original LifeAct construct (RRID: Addgene_58467) with the red-fluorescent mCherry, resulting in cells showing a homogenous nuclear LifeAct signal which was slightly enriched in what seemed to be nucleoli.

Techniques: Infection, Staining

Journal: bioRxiv

Article Title: Human cytomegalovirus nuclear capsid motility is non-directed and independent of nuclear actin bundles

doi: 10.1101/641266

Figure Lengend Snippet: BJ-CMV-LifeAct-mCherry, WT BJ and BJ-CMV-LifeAct-mCherry-NLS were infected with HCMV-HB5-UL77-mGFP at an MOI of 10 for 24 hours, fixed, and stained for IE1, as well as with Alexa-488-Phalloidin. (A) No nuclear LifeAct-mCherry-stainable structures were observed without the NLS. For comparison, infected WT-BJ cells stained with Alexa-488-Phalloidin, and BJ-CMV-LifeAct-mCherry-NLS are shown. Scale bars indicate 10 µm.

Article Snippet: Since this UL77-mGFP fusion already occupied the GFP channel, we exchanged GFP in the original LifeAct construct (RRID: Addgene_58467) with the red-fluorescent mCherry, resulting in cells showing a homogenous nuclear LifeAct signal which was slightly enriched in what seemed to be nucleoli.

Techniques: Infection, Staining

Journal: bioRxiv

Article Title: Human cytomegalovirus nuclear capsid motility is non-directed and independent of nuclear actin bundles

doi: 10.1101/641266

Figure Lengend Snippet: BJ-CMV-LifeAct-mCherry-NLS cells were infected with HCMV-HB5-UL77-mGFP and imaged live at 72hpi at a frame rate of 21.45 fps. A maximum temporal projection of the GFP channel over 600 frames shows diffusive green clouds of particle location. The video is available as supplementary video S1. Scale bar indicates 10 µm.

Article Snippet: Since this UL77-mGFP fusion already occupied the GFP channel, we exchanged GFP in the original LifeAct construct (RRID: Addgene_58467) with the red-fluorescent mCherry, resulting in cells showing a homogenous nuclear LifeAct signal which was slightly enriched in what seemed to be nucleoli.

Techniques: Infection

Journal: bioRxiv

Article Title: Human cytomegalovirus nuclear capsid motility is non-directed and independent of nuclear actin bundles

doi: 10.1101/641266

Figure Lengend Snippet: BJ or BJ-CMV-LifeAct-mCherry-NLS cells were infected with HCMV-TB40/e-UL32EGFP-UL100mCherry, high-pressure-frozen at 24hpi and freeze-substituted. (A) Bundled, filamentous actin structures can be visualized in the nuclei of infected cells expressing LifeAct-mCherry-NLS. Bundles only appeared in a fraction of the examined cells and in higher frequency closer to the nuclear envelope (see also supplementary Figure S3). (B) Two cells without apparent nuclear actin structures visible for comparison. For scale bar length see picture.

Article Snippet: Since this UL77-mGFP fusion already occupied the GFP channel, we exchanged GFP in the original LifeAct construct (RRID: Addgene_58467) with the red-fluorescent mCherry, resulting in cells showing a homogenous nuclear LifeAct signal which was slightly enriched in what seemed to be nucleoli.

Techniques: Infection, Expressing

Journal: bioRxiv

Article Title: Human cytomegalovirus nuclear capsid motility is non-directed and independent of nuclear actin bundles

doi: 10.1101/641266

Figure Lengend Snippet: BJ-SFFV-LifeAct-mCherry-NLS cells were infected with HCMV-TB40/e-UL32EGFP-UL100mCherry, fixed at 24hpi and stained for DNA (Hoechst) and HCMV-IE (Anti-IE1). (A) Representative image of very rare nuclear actin filaments. Scale bar indicates 10 µm. (B) Quantification of the filament rates. Large tiles spanning 0.75×0.75 µm were acquired and quantified in ImageJ and Python using scripts (see Materials and Methods). Filaments were manually counted. Means of three independent replicates are shown. Bars indicate standard deviations.

Article Snippet: The lentiviral plasmid LeGO_SFFV_LifeAct-mCh-2XNLS was generated by designing a LifeAct-mCherry-2XNLS insert reflecting the pEGFP-C1_LifeAct-EGFP_2XNLS construct ( ). (pEGFP-C1 LifeAct-EGFP-2XNLS was a gift from Dyche Mullins (RRID: Addgene_58467).

Techniques: Infection, Staining

Journal: bioRxiv

Article Title: Human cytomegalovirus nuclear capsid motility is non-directed and independent of nuclear actin bundles

doi: 10.1101/641266

Figure Lengend Snippet: BJ-SFFV-Life-Act-mCherry-NLS cells were infected with HCMV-TB40/e-UL32EGFP-UL100mCherry, fixed at the indicated time points and stained for DNA (Hoechst) and HCMV-IE (Anti-IE1). pUL32-EGFP serves as a marker for late gene expression. (A) LifeAct-mCherry-NLS signal intensity drops with ongoing infection. Scale bars indicate 10 µm. (B) Quantification of subcellular Life-Act vs. IE signal intensities at 24, 48 and 72hpi using automated microscopy image analysis. (C) LifeAct intensity over time. For comparison, all image intensities are scaled to the same level in and . Scale bars indicate 10 µm.

Article Snippet: The lentiviral plasmid LeGO_SFFV_LifeAct-mCh-2XNLS was generated by designing a LifeAct-mCherry-2XNLS insert reflecting the pEGFP-C1_LifeAct-EGFP_2XNLS construct ( ). (pEGFP-C1 LifeAct-EGFP-2XNLS was a gift from Dyche Mullins (RRID: Addgene_58467).

Techniques: Infection, Staining, Marker, Expressing, Microscopy

Journal: bioRxiv

Article Title: Human cytomegalovirus nuclear capsid motility is non-directed and independent of nuclear actin bundles

doi: 10.1101/641266

Figure Lengend Snippet: BJ-CMV-LifeAct-mCherry-NLS cells were infected with HCMV-TB40/e-UL32EGFP-UL100mCherry, fixed at the indicated time points and stained for DNA (Hoechst) and HCMV-IE (Anti-IE1). pUL32-EGFP serves as a marker for late gene expression. (A) LifeAct-mCherry-NLS signal intensity drops with ongoing infection. Scale bars indicate 10 µm. (B) Quantification of subcellular LifeAct vs. IE signal intensities at 24, 48 and 72hpi by automated microscopy image analysis. (C) LifeAct intensity over time. All image intensities are scaled to the same level in and for comparison. Scale bars indicate 10 µm.

Article Snippet: The lentiviral plasmid LeGO_SFFV_LifeAct-mCh-2XNLS was generated by designing a LifeAct-mCherry-2XNLS insert reflecting the pEGFP-C1_LifeAct-EGFP_2XNLS construct ( ). (pEGFP-C1 LifeAct-EGFP-2XNLS was a gift from Dyche Mullins (RRID: Addgene_58467).

Techniques: Infection, Staining, Marker, Expressing, Microscopy

Journal: bioRxiv

Article Title: Human cytomegalovirus nuclear capsid motility is non-directed and independent of nuclear actin bundles

doi: 10.1101/641266

Figure Lengend Snippet: BJ-CMV-LifeAct-mCherry-NLS cells were infected with HCMV-TB40/e-UL32EGFP-UL100mCherry, fixed at the indicated time points and stained for DNA (Hoechst) and HCMV-IE (Anti-IE1). (A-D) Rate of LifeAct nuclear filaments in infected IE-1-positive cells. Four replicates with different base-line expression levels of LifeAct are shown as quantified in (E). (F) Difference in LifeAct-mCherry-NLS signal intensity in the cells with filaments (+), compared to those without (-).

Article Snippet: The lentiviral plasmid LeGO_SFFV_LifeAct-mCh-2XNLS was generated by designing a LifeAct-mCherry-2XNLS insert reflecting the pEGFP-C1_LifeAct-EGFP_2XNLS construct ( ). (pEGFP-C1 LifeAct-EGFP-2XNLS was a gift from Dyche Mullins (RRID: Addgene_58467).

Techniques: Infection, Staining, Expressing

Journal: bioRxiv

Article Title: Human cytomegalovirus nuclear capsid motility is non-directed and independent of nuclear actin bundles

doi: 10.1101/641266

Figure Lengend Snippet: BJ-CMV-LifeAct-mCherry-NLS cells were infected with HCMV-TB40/e-UL32EGFP-UL100mCherry, fixed at 72hpi and stained for DNA (Hoechst) and HCMV-IE (Anti-IE1). pUL32-EGFP serves as a marker for late gene expression. (A ) Cells that show pUL32-EGFP expression have very little LifeAct-mCherry-NLS signal. Scale bar indicates 20 µm. (B) Scatter plot of nuclear LifeAct-mCherry-NLS vs. pUL32-EGFP signal compared to the nuclear mCherry signal.

Article Snippet: The lentiviral plasmid LeGO_SFFV_LifeAct-mCh-2XNLS was generated by designing a LifeAct-mCherry-2XNLS insert reflecting the pEGFP-C1_LifeAct-EGFP_2XNLS construct ( ). (pEGFP-C1 LifeAct-EGFP-2XNLS was a gift from Dyche Mullins (RRID: Addgene_58467).

Techniques: Infection, Staining, Marker, Expressing

Journal: bioRxiv

Article Title: Human cytomegalovirus nuclear capsid motility is non-directed and independent of nuclear actin bundles

doi: 10.1101/641266

Figure Lengend Snippet: WT BJ and BJ-CMV-LifeAct-mCherry-NLS were infected with HCMV-HB5-UL77-mGFP at an MOI of 10 for 24 hours, fixed, and stained for IE1, as well as with Alexa-488-Phalloidin. The arrow indicates a representative BJ-CMV-LifeAct-mCherry-NLS cell in which the same nuclear actin structures are stained by both LifeAct as well as by Phalloidin. Scale bar is 10 µm.

Article Snippet: The lentiviral plasmid LeGO_SFFV_LifeAct-mCh-2XNLS was generated by designing a LifeAct-mCherry-2XNLS insert reflecting the pEGFP-C1_LifeAct-EGFP_2XNLS construct ( ). (pEGFP-C1 LifeAct-EGFP-2XNLS was a gift from Dyche Mullins (RRID: Addgene_58467).

Techniques: Infection, Staining

Journal: bioRxiv

Article Title: Human cytomegalovirus nuclear capsid motility is non-directed and independent of nuclear actin bundles

doi: 10.1101/641266

Figure Lengend Snippet: BJ-CMV-LifeAct-mCherry, WT BJ and BJ-CMV-LifeAct-mCherry-NLS were infected with HCMV-HB5-UL77-mGFP at an MOI of 10 for 24 hours, fixed, and stained for IE1, as well as with Alexa-488-Phalloidin. (A) No nuclear LifeAct-mCherry-stainable structures were observed without the NLS. For comparison, infected WT-BJ cells stained with Alexa-488-Phalloidin, and BJ-CMV-LifeAct-mCherry-NLS are shown. Scale bars indicate 10 µm.

Article Snippet: The lentiviral plasmid LeGO_SFFV_LifeAct-mCh-2XNLS was generated by designing a LifeAct-mCherry-2XNLS insert reflecting the pEGFP-C1_LifeAct-EGFP_2XNLS construct ( ). (pEGFP-C1 LifeAct-EGFP-2XNLS was a gift from Dyche Mullins (RRID: Addgene_58467).

Techniques: Infection, Staining

Journal: bioRxiv

Article Title: Human cytomegalovirus nuclear capsid motility is non-directed and independent of nuclear actin bundles

doi: 10.1101/641266

Figure Lengend Snippet: BJ-CMV-LifeAct-mCherry-NLS cells were infected with HCMV-HB5-UL77-mGFP and imaged live at 72hpi at a frame rate of 21.45 fps. A maximum temporal projection of the GFP channel over 600 frames shows diffusive green clouds of particle location. The video is available as supplementary video S1. Scale bar indicates 10 µm.

Article Snippet: The lentiviral plasmid LeGO_SFFV_LifeAct-mCh-2XNLS was generated by designing a LifeAct-mCherry-2XNLS insert reflecting the pEGFP-C1_LifeAct-EGFP_2XNLS construct ( ). (pEGFP-C1 LifeAct-EGFP-2XNLS was a gift from Dyche Mullins (RRID: Addgene_58467).

Techniques: Infection

Journal: bioRxiv

Article Title: Human cytomegalovirus nuclear capsid motility is non-directed and independent of nuclear actin bundles

doi: 10.1101/641266

Figure Lengend Snippet: BJ or BJ-CMV-LifeAct-mCherry-NLS cells were infected with HCMV-TB40/e-UL32EGFP-UL100mCherry, high-pressure-frozen at 24hpi and freeze-substituted. (A) Bundled, filamentous actin structures can be visualized in the nuclei of infected cells expressing LifeAct-mCherry-NLS. Bundles only appeared in a fraction of the examined cells and in higher frequency closer to the nuclear envelope (see also supplementary Figure S3). (B) Two cells without apparent nuclear actin structures visible for comparison. For scale bar length see picture.

Article Snippet: The lentiviral plasmid LeGO_SFFV_LifeAct-mCh-2XNLS was generated by designing a LifeAct-mCherry-2XNLS insert reflecting the pEGFP-C1_LifeAct-EGFP_2XNLS construct ( ). (pEGFP-C1 LifeAct-EGFP-2XNLS was a gift from Dyche Mullins (RRID: Addgene_58467).

Techniques: Infection, Expressing