plasmid pegfp c1 lifeact egfp 2xnls  (Addgene inc)

Journal: Advanced Science

Article Title: Low YTHDC1 Expression Upregulates FSCN1 to Promote Nuclear F‐Actin Formation and Facilitate Double‐strand DNA Breaks Repair in TMZ‐Resistant Glioblastoma

doi: 10.1002/advs.202513632

Figure Lengend Snippet: Nuclear F‐actin increase and promote TMZ resistance in GBM cells. (A) Immunofluorescence analysis of GBM cells stained with phalloidin, anti‐Lamin B1 antibody and Lifeact‐EGFP‐2xNLS plasmids. Scale bar = 5µm. (B) Normalized timecourse of pyrene‐labelled actin assembly in the LN229, 229R, HG7 and HG7R nuclear extracts. (C) Western Blot (top) and analysis of globular (G‐) and filamentous (F‐) actin indicate the rate of actin polymerization in GBM cells (n = 3). (D) Immunofluorescence analysis of GBM cells treated with Lifeact‐EGFP‐2xNLS plasmids. Scale bar = 5µm. (E) Normalized timecourse of pyrene‐labelled actin assembly in the TMZ‐resistant GBM cells nuclear extracts after DMSO or LatB treatment. (F) Western Blot (top) and analysis of globular (G‐) and filamentous (F‐) actin indicate the rate of actin polymerization in TMZ‐resistant cells after DMSO or LatB treatment (n = 3). (G) Immunofluorescence analysis of TMZ‐resistant GBM cells treated with Lifeact‐EGFP‐2xNLS plasmids after DMSO or LatB treatment. Scale bar = 5µm. (H) Flow cytometric analysis revealed the effect of F‐actin suppressed on the apoptosis of TMZ‐resistant cells with or without TMZ treatment (n = 3). (I) Colony formation assay detected the effect of F‐actin suppressed on the growth of TMZ‐resistant cells with or without TMZ treatment in a 6‐well dish (800 cells per well) for 11 days (n = 3). Representative images and the relative number of colonies are shown. For A, D and G, scale bars, 5 µm. Data were analyzed using Student's t‐test (C, F, H and I). Significant results were presented as NS non‐significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The plasmid pEGFP‐C1 Lifeact‐EGFP‐2XNLS (Addgene, USA) was sourced from Addgene.

Techniques: Immunofluorescence, Staining, Western Blot, Colony Assay

Journal: Advanced Science

Article Title: Low YTHDC1 Expression Upregulates FSCN1 to Promote Nuclear F‐Actin Formation and Facilitate Double‐strand DNA Breaks Repair in TMZ‐Resistant Glioblastoma

doi: 10.1002/advs.202513632

Figure Lengend Snippet: FSCN1 regulates nuclear F‐actin formation to promote DNA damage repair. (A) Immunofluorescence analysis of γH2AX in TMZ‐resistant GBM cells treated with DMSO, TMZ or LatB. The nuclei were stained with DAPI. Quantification of γH2AX in Image J. Scale bar = 5µm. (B) Comet assays measuring the DNA damage degree in 229R cells treated with DMSO, LatB, TMZ and TMZ + LatB. Scale bar = 100µm. Comet tails were measured by image j. (C) Western blot analysis of p‐ATM and γH2AX expression in TMZ‐resistant GBM cells treated with DMSO, TMZ or LatB. (D) Western blot analysis of FSCN1 expression in parental and TMZ‐resistant GBM cell nucleus (top) and western blot analysis of FSCN1 expression in parental and TMZ‐resistant GBM cell (bottom). (E) Western blot analysis of FSCN1 expression in paired primary and recurrent tissue cell nucleus from clinical patients (top) and western blot analysis of FSCN1 expression in paired primary and recurrent tissue cell from clinical patients (bottom). (F) Immunofluorescence analysis of FSCN1 expression in parental and TMZ‐resistant GBM cells. Scale bar = 50µm. (G) Western blot analysis of FSCN1 expression in FSCN1 knockdown TMZ‐resistant GBM cell nucleus (left) and western blot analysis of FSCN1 expression in FSCN1 knockdown TMZ‐resistant GBM cells (right). (H) Immunofluorescence analysis of FSCN1 knockdown TMZ‐resistant GBM cells treated with Lifeact‐EGFP‐2xNLS plasmids. Scale bar = 5µm. (I) Comet assays measuring the DNA damage degree in shNC 229R cells treated with DMSO, TMZ and shFSCN1 229R cells treated with DMSO and TMZ. (J) Western blot analysis of p‐ATM and γH2AX expression in shFSCN1 229R cells treated with or without TMZ. For A, and H, scale bars, 5 µm. For F, scale bars, 50 µm. For B, scale bars, 100 µm. Data were analyzed using Student's t‐test (B and I). Significant results were presented as NS non‐significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The plasmid pEGFP‐C1 Lifeact‐EGFP‐2XNLS (Addgene, USA) was sourced from Addgene.

Techniques: Immunofluorescence, Staining, Western Blot, Expressing, Knockdown

Journal: Advanced Science

Article Title: Low YTHDC1 Expression Upregulates FSCN1 to Promote Nuclear F‐Actin Formation and Facilitate Double‐strand DNA Breaks Repair in TMZ‐Resistant Glioblastoma

doi: 10.1002/advs.202513632

Figure Lengend Snippet: YTHDC1 downregulation in TMZ‐resistant GBM cells and recurrent patients correlates with TMZ resistance. (A) Relative expression of YTHDC1 in primary (n = 27) and recurrent (n = 11) GBM sample tissues. (B) Relative expression of YTHDC1 mRNA in parental and TMZ‐resistant GBM cells (n = 3). (C) Western blot analysis of YTHDC1 expression in paired primary and recurrent tissues from clinical patients. (D) Western blot analysis of YTHDC1 expression in parental and TMZ‐resistant GBM cells. (E) IHC staining of YTHDC1 in paired primary and recurrent tissues from clinical patients. Scale bar = 200µm. (F) Immunofluorescence analysis of YTHDC1 expression in parental and TMZ‐resistant GBM cells. The nuclei were stained with DAPI. Cytoskeletal staining with phalloidin. Scale bar = 50µm. (G) Immunofluorescence analysis of YTHDC1 expression in GBM cells treated with Lifeact‐EGFP‐2xNLS plasmids. Scale bar = 5µm. (H) Flow cytometric analysis revealed the apoptosis of YTHDC1 knockdown parental GBM cells and YTHDC1 overexpression TMZ‐resistant GBM cells with TMZ treatment (n = 3). (I) Colony formation assay detected the growth of YTHDC1 knockdown parental GBM cells and YTHDC1 overexpression TMZ‐resistant GBM cells with TMZ treatment in a 6‐well dish (800 cells per well) for 11 days (n = 3). (J) CCK‐8 assay analysis revealed the effect of YTHDC1 knockdown in parental GBM cells and YTHDC1 overexpression TMZ‐resistant GBM cells with TMZ treatment at the indicated concentrations for 72 h (n = 3). (K) Western blot analysis of FSCN1 rescue experiment in LN229 shYTHDC1 cells with shFSCN1 and in 229R OE‐YTHDC1 cells with OE‐FSCN1. (L) Immunofluorescence with Lifeact‐EGFP‐2xNLS plasmids treatment analysis of FSCN1 rescue experiment in LN229 shYTHDC1 cells with shFSCN1 and in 229R OE‐YTHDC1 cells with OE‐FSCN1. Scale bar = 5µm. (M) Flow cytometric analysis of FSCN1 rescue experiment in LN229 shYTHDC1 cells with shFSCN1 and in 229R OE‐YTHDC1 cells with OE‐FSCN1 (n = 3). For G and L, scale bars, 5 µm. For F, scale bars, 50 µm. For E, scale bars, 200 µm. Data were analyzed using Student's t‐test (A, B, H, I and M) and four‐parameter logistic regression (J). Significant results were presented as NS non‐significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The plasmid pEGFP‐C1 Lifeact‐EGFP‐2XNLS (Addgene, USA) was sourced from Addgene.

Techniques: Expressing, Western Blot, Immunohistochemistry, Immunofluorescence, Staining, Knockdown, Over Expression, Colony Assay, CCK-8 Assay

Journal: Advanced Science

Article Title: Low YTHDC1 Expression Upregulates FSCN1 to Promote Nuclear F‐Actin Formation and Facilitate Double‐strand DNA Breaks Repair in TMZ‐Resistant Glioblastoma

doi: 10.1002/advs.202513632

Figure Lengend Snippet: FSCN1 activates the CDC42/N‐WASP/Arp2/3 axis by recruiting FGD1. (A) Western blot analysis of CDC42 after pulldown by PAK‐PBD in the nucleus of LN229 and 229R cells. (B) Left: Western blot analysis in the nucleus of LN229 and 229R GBM cells with N‐WASP antibody, Arp2 antibody and Lamin B1 antibody. Right: Immunoprecipitation in the nucleus of LN229 and 229R GBM cells with an antibody against N‐WASP; the cells were subsequently labeled with the anti‐Arp2 antibody. (C) Western blot analysis of CDC42 after pulldown by PAK‐PBD in the nucleus of 229R shNC and 229R shFSCN1 GBM cells. (D) Upper: Western blot analysis in the nucleus of 229R shNC and 229R shFSCN1 GBM cells with N‐WASP antibody, Arp2 antibody and Lamin B1 antibody. Lower: Immunoprecipitation in the nucleus of 229R shNC and 229R shFSCN1 cells with an antibody against N‐WASP; the cells were subsequently labeled with the anti‐Arp2 antibody. (E) Normalized timecourse of pyrene‐labelled actin assembly in 229R shNC and 229R shFSCN1 cell nuclear extracts. (F) Western blot analysis of FGD1 in parental and TMZ‐resistant GBM cell nuclear extracts. (G) Western blot analysis of FGD1 in 229R shNC and 229R shFSCN1 GBM cell nuclear extracts. (H) Upper: Immunoprecipitation of 229R GBM cells with an antibody against FSCN1; the cells were subsequently labeled with the anti‐FGD1 antibody. Lower: Immunoprecipitation of 229R GBM cells with an antibody against FGD1; the cells were subsequently labeled with the anti‐FSCN1 antibody. (I) Western blot analysis in the nucleus of LN229 OE‐NC and LN229 OE‐FSCN1 GBM cells with FSCN1 antibody, FGD1 antibody and Lamin B1 antibody. (J) Normalized timecourse of pyrene‐labelled actin assembly in LN229 OE‐NC and LN229 OE‐FSCN1 GBM cell nuclear extracts. (K) Western blot analysis in the nucleus of LN229 OE‐FSCN1 shNC and LN229 OE‐FSCN1 shFGD1 GBM cells with FSCN1 antibody, FGD1 antibody and Lamin B1 antibody. (L) Normalized timecourse of pyrene‐labelled actin assembly in LN229 OE‐FSCN1 shNC and LN229 OE‐FSCN1 shFGD1 GBM cell nuclear extracts. (M) Immunofluorescence analysis of 229R cells treated with Lifeact‐EGFP‐2xNLS plasmids after treatment with DMSO or ML141 (10 µM, 37°C, 1 h). Scale bar = 5µm. (N) Immunofluorescence analysis of 229R cells treated with Lifeact‐EGFP‐2xNLS plasmids after treatment with DMSO or CK‐666 (100 µM, 37°C, 6 h). Scale bar = 5µm. (O) Immunofluorescence of γH2AX foci in FSCN1 knockdown TMZ‐resistant GBM cells with TMZ treatment show γH2AX foci in DAPIbright heterochromatin. Scale bar = 5µm. (P) Immunofluorescence of γH2AX foci in TMZ‐resistant GBM cells with TMZ or TMZ + ML141 (10 µM, 37°C, 1 h) treatment show γH2AX foci in DAPIbright heterochromatin. Scale bar = 5µm. (Q) Immunofluorescence of γH2AX foci in TMZ‐resistant GBM cells with TMZ or TMZ + CK‐666 (100 µM, 37°C, 6 h) treatment show γH2AX foci in DAPIbright heterochromatin. Scale bar = 5µm. (R) Detection of HR activity using the DR‐GFP reporter system. Different combinations of DR‐GFP reporter plasmids were co‐transfected into LN229 shNC and shFSCN1 cells, as well as 229R OE‐NC and OE‐FSCN1 cells. Cells were treated with 100µM TMZ for 24 h, and the percentage of GFP‐positive cells was determined by flow cytometry (n = 3). For M, N, O, P and Q scale bars, 5µm. Data were analyzed using Student's t‐test (R). Significant results were presented as NS non‐significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The plasmid pEGFP‐C1 Lifeact‐EGFP‐2XNLS (Addgene, USA) was sourced from Addgene.

Techniques: Western Blot, Immunoprecipitation, Labeling, Immunofluorescence, Knockdown, Activity Assay, Transfection, Flow Cytometry

Journal: Advanced Science

Article Title: Low YTHDC1 Expression Upregulates FSCN1 to Promote Nuclear F‐Actin Formation and Facilitate Double‐strand DNA Breaks Repair in TMZ‐Resistant Glioblastoma

doi: 10.1002/advs.202513632

Figure Lengend Snippet: Treatment with TMZ combined with the FSCN1 inhibitor NP‐G2‐044 reverses GBM TMZ resistance both in vitro and in vivo. (A) After treatment at different temperatures ranging from 37°C to 72°C, proteins were extracted and analyzed by Western blot for FSCN1 and GAPDH. DMSO treatment served as the control group, and NP‐G2‐044 treatment as the experimental group (left). The band intensity was quantified to compare the thermal stability of FSCN1 at different temperatures (right, n = 3). (B) Immunofluorescence analysis of GBM cells treated with Lifeact‐EGFP‐2xNLS plasmids after treatment with DMSO or NP‐G2‐044. Scale bar = 5µm. (C) Immunofluorescence analysis of GBM cells stained with γH2AX antibody treated with TMZ or NP‐G2‐044. The nuclei were stained with DAPI. Quantification of γH2AX in Image J (n = 3). Scale bar = 5µm. (D) Western blot analysis of p‐ATM and γH2AX expression in TMZ‐resistant GBM cells treated with TMZ or NP‐G2‐044. E CCK‐8 assay analysis revealed the effect of TMZ‐resistant GBM cells with TMZ or NP‐G2‐044 treatment at the indicated concentrations for 72 h (n = 3). F Nude mice were orthotopically xenografted with 229R GBM cells (5 × 10 5 cells) and treated intraperitoneally with TMZ (60 mg kg −1 day −1 per mouse) or intratumorally with NP‐G2‐044 (100 mg kg −1 day −1 per mouse) every 5 days. G Bioluminescent images of nude mice. (H) Quantification of bioluminescent imaging signal intensities in nude mice. (I) Kaplan‐Meier survival curve of nude mice is shown. For B and C, scale bars, 5 µm. Data were analyzed using Student's t ‐test (A, E and H) and four‐parameter logistic regression (E) and Log Rank test (I). Significant results were presented as NS non‐significant, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: The plasmid pEGFP‐C1 Lifeact‐EGFP‐2XNLS (Addgene, USA) was sourced from Addgene.

Techniques: In Vitro, In Vivo, Western Blot, Control, Immunofluorescence, Staining, Expressing, CCK-8 Assay, Imaging

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

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