independent shrna constructs  (OriGene)

Journal: bioRxiv

Article Title: Loss of Sun2 ablates nuclear mechanosensing-driven extracellular matrix production and mitigates lung fibrosis

doi: 10.64898/2026.03.18.712778

Figure Lengend Snippet: ( A ) Immunofluorescence staining of alpha-smooth muscle actin (αSMA) and SUN2 in lung tissue samples from healthy human patients and human patients with idiopathic lung fibrosis (IPF). ( B ) SUN2 immunofluorescence staining intensity at the nuclear envelope in healthy and IPF patient lung tissue was quantified in ImageJ, averaging the intensity of >3 fields of view per patient, N=5 patients per condition. No significant difference was found, determined by unpaired two-tailed t test; error bars are SD. ( C ) αSMA+/- cells in IPF patient tissue were identified using QuPath. SUN2 immunofluorescence staining intensity was then measured in both αSMA+/- cells, averaging intensity at the nuclear envelope in cells from >3 fields of view per patient, N=5 patients. There was a significantly higher intensity of SUN2 immunofluorescence staining at the nuclear envelope of αSMA+ cells when compared to αSMA- cells within IPF patient lung tissue. Statistical significance was determined by unpaired two-tailed t test; ***p < 0.001; error bars are SD.

Article Snippet: Sun2 -/- (strain B6;129S6-Sun2tm1Mhan/J) and C57BL/6 WT mice were obtained from Jackson Laboratories.

Techniques: Immunofluorescence, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Loss of Sun2 ablates nuclear mechanosensing-driven extracellular matrix production and mitigates lung fibrosis

doi: 10.64898/2026.03.18.712778

Figure Lengend Snippet: ( A ) Schematic demonstrating the time course of bleomycin inhalation induced fibrosis. ( B ) Immunofluorescence staining of Sun2 in wildtype mouse lung tissue 2 days post bleomycin inhalation and 14 days post bleomycin inhalation. ( C ) Sun2 nuclear staining intensity is significantly increased at day 14 post bleomycin inhalation during peak fibrosis when compared to day 2 post bleomycin inhalation. N=3 mice per condition. >2 fields of view analyzed for each mouse. Statistical significance was determined by unpaired two-tailed t test; **p < 0.01; error bars are SD. ( D-E ) Immunofluorescence staining of Nesprin-1 in wildtype mouse lung tissue 2 days and 14 days post bleomycin inhalation. Nesprin-1 nuclear staining intensity is significantly increased at day 14 post bleomycin inhalation during peak fibrosis when compared to day 2 post bleomycin inhalation. N=3 mice per condition, >3 fields of view analyzed for each mouse. Statistical significance was determined by unpaired two-tailed t test; ***p < 0.001; error bars are SD. ( F ) Dot plot of Sun2 , Syne1 (encoding Nesprin-1), secretory fibroblast marker Cthrc1 , and collagens Col1a1 and Col3a1 expression in the fibrotic fibroblast cluster of Col1-GFP expressing cells (Supplemental Fig. S2A) from scRNA-seq data in mouse lungs 7, 14, and 21 days after bleomycin inhalation. Data from GEO: GSE210341. ( G ) Gene Ontology (GO) biological process analysis of genes differentially upregulated in cells with high Sun2 expression compared to those with low Sun2 expression within the fibrotic cluster.

Article Snippet: Sun2 -/- (strain B6;129S6-Sun2tm1Mhan/J) and C57BL/6 WT mice were obtained from Jackson Laboratories.

Techniques: Immunofluorescence, Staining, Two Tailed Test, Marker, Expressing

Journal: bioRxiv

Article Title: Loss of Sun2 ablates nuclear mechanosensing-driven extracellular matrix production and mitigates lung fibrosis

doi: 10.64898/2026.03.18.712778

Figure Lengend Snippet: ( A ) Immunofluorescence staining of Sun2 in primary wildtype lung fibroblasts seeded on glass and 2 kPa Matrigen substrates for 24 hours. ( B ) Superplot of Sun2 nuclear staining intensity, which is significantly increased in fibroblasts plated on glass compared to a 2 kPa substrate. n=>100 cells, N=3 replicates. Statistical significance was determined by unpaired two-tailed t test; ****p < 0.0001; error bars are SD. ( C ) Plot showing fold change in mRNA expression of Sun2 in primary wildtype lung fibroblasts cultured on 2 kPa versus glass substrates. There is no significant difference in Sun2 expression between substrate stiffnesses as determined by two-tailed t test. N=3 replicates. Error bars are SD.

Article Snippet: Sun2 -/- (strain B6;129S6-Sun2tm1Mhan/J) and C57BL/6 WT mice were obtained from Jackson Laboratories.

Techniques: Immunofluorescence, Staining, Two Tailed Test, Expressing, Cell Culture

Journal: bioRxiv

Article Title: Loss of Sun2 ablates nuclear mechanosensing-driven extracellular matrix production and mitigates lung fibrosis

doi: 10.64898/2026.03.18.712778

Figure Lengend Snippet: ( A ) Loss of Sun2 showed reduced collagen as assessed by Sircol assay at 14 and 21-days post-bleomycin administration. N=>4 mice per condition, male and female mice. Statistical significance was determined by performing multiple t-tests; * p<0.05, ** p<0.01. ( B ) Trichrome staining to visualize collagen (blue) was analyzed to determine disease severity using a ( C ) Modified Ashcroft Score, showing less disease severity in Sun2 -/- mice. N=10 mice per genotype, includes both male and female mice. Statistical significance was determined by performing a two-tailed Student’s t test; * p<0.05; Error bars are SD.

Article Snippet: Sun2 -/- (strain B6;129S6-Sun2tm1Mhan/J) and C57BL/6 WT mice were obtained from Jackson Laboratories.

Techniques: Staining, Modification, Two Tailed Test

Journal: bioRxiv

Article Title: Loss of Sun2 ablates nuclear mechanosensing-driven extracellular matrix production and mitigates lung fibrosis

doi: 10.64898/2026.03.18.712778

Figure Lengend Snippet: ( A ) Apoptotic cells were stained using TUNEL and the percentage of apoptotic cells were quantified for tissue 2 days post-bleomycin administration. N=6 male and female mice. There is no statistically significant difference (ns) in these levels upon loss of Sun2 based on a Student’s t test. ( B ) The number of cells present in the BAL is not impacted by the loss of Sun2 during the inflammatory response phase of the model (prior to Day 14). N=>4 mice per condition, male and female mice. Statistical significance was determined by performing multiple t-tests; ns no significance; * = p<0.05. The Holm–Sidak method was used to correct for multiple comparisons. Error bars are SD. ( C ) Sun2 is dispensable for the recovery of the alveolar barrier as assessed by the kinetics of the increase and subsequent decrease in albumin leaking from the blood into the BAL. ( D ) Wildtype and Sun2 -/- mouse lung tissue from mice euthanized 14 days post bleomycin inhalation were stained with an antibody against phospho-Smad2/3. ( E ) Superplot of the ratio of nuclear to cytoplasmic levels of pSmad2/3 in fibrotic mouse lung tissue shows a significant increase in pSmad2/3 translocation to the nucleus in Sun2 -/- tissue during peak fibrosis. N=3 male mice per genotype, >1 field of view analyzed per mouse. Determined by two-tailed unpaired t test, ****p < 0.0001. Data from three replicates superimposed in blue, orange, and pink. ( F ) Immunofluorescence staining of lung tissue sections from mice 2- or 14-days post-bleomycin administration was performed using an antibody against alpha smooth muscle actin (αSMA) to visualize hypercontractile cells. Regions marked by the dashed box are shown at higher magnification below. ( G ) There is an expected increase in αSMA positive tissue over time but no significant difference in αSMA positive cell levels in WT and Sun2 -/- tissue, as was determined by an ordinary one-way ANOVA followed by Sidak’s multiple comparisons test. Error bars are SD. ( H ) There is no significant difference between the size of the gel contracted by wildtype lung fibroblasts and Sun2 -/- fibroblasts after 24 hours as determined by two-tailed unpaired t-test. N=6 replicates. Error bars are SD.

Article Snippet: Sun2 -/- (strain B6;129S6-Sun2tm1Mhan/J) and C57BL/6 WT mice were obtained from Jackson Laboratories.

Techniques: Staining, TUNEL Assay, Translocation Assay, Two Tailed Test, Immunofluorescence

Journal: bioRxiv

Article Title: Loss of Sun2 ablates nuclear mechanosensing-driven extracellular matrix production and mitigates lung fibrosis

doi: 10.64898/2026.03.18.712778

Figure Lengend Snippet: ( A ) Heat map showing expression patterns of genes characteristic of fibrotic fibroblasts in primary wildtype and Sun2 -/- fibroblasts cultured on 50 kPa in the presence and absence of exogenous TGFβ1 (5ng/ml)(GEO: GSE325284). Overall, compared to WT, Sun2 -/- fibroblasts exhibit increased expression of Cthrc1 , and decreased expression of extracellular matrix genes in the absence or presence of TGFβ1. ( B ) Immunostaining of pro-collagen in primary lung fibroblasts isolated from wildtype and Sun2 -/- mice after growing on glass coverslips for 24 hours. ( C ) Less Sun2 -/- fibroblasts produce pro-collagen. Pro-collagen positivity was determined by the automatic thresholding feature on Fiji. The number of cells positive for pro-collagen staining was counted for each genotype and divided by the total number of cells per replicate and multiplied by 100 to determine a percentage. n=50< cells, N=3 replicates. Determined by two-tailed unpaired t test; ***p < 0.001. ( D ) Cartoon diagram showing our working model in which Sun2 acts as a component of a key nuclear mechanotransduction cascade coupling myofibroblast contractility to collagen production in response to lung injury. In response to injury, subsets of fibroblasts express organized αSMA fibers, contracting surrounding tissue and altering the mechanical environment in both wildtype and Sun2 -/- tissue. Mechanical signals transduced via Sun2-containing LINC complexes in αSMA+ and/or αSMA-fibroblasts prime an increase in transcription of ECM genes. This nuclear mechanosensation is disrupted in Sun2 -/- fibroblasts, which fail to produce pathogenic ECM.

Article Snippet: Sun2 -/- (strain B6;129S6-Sun2tm1Mhan/J) and C57BL/6 WT mice were obtained from Jackson Laboratories.

Techniques: Expressing, Cell Culture, Immunostaining, Isolation, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Loss of Sun2 ablates nuclear mechanosensing-driven extracellular matrix production and mitigates lung fibrosis

doi: 10.64898/2026.03.18.712778

Figure Lengend Snippet: Cartoon diagram showing our working model in which Sun2 acts as a component of a key nuclear mechanotransduction cascade coupling a stiffening tissue microenvironment to new collagen deposition in response to lung injury. In the absence of injury, integrins are not strongly engaged, the cytoskeleton is more relaxed, and there is low tension on LINC complexes and the nucleus, corresponding to repression of TGFβ targets including ECM genes and α-SMA. In response to injury, TGFβ binds to the TGFβ receptor and phosphorylated Smads translocate to the nucleus, representing the canonical biochemical cascade. In addition, tension from integrins is propagated through the actin cytoskeleton to LINC complexes. LINC complexes are up-regulated, inducing high tension on the nucleus. Genes contributing to pathogenic ECM production and contractility (ECM genes and α-SMA) are induced, with ECM genes requiring coincidence detection of both nuclear tension and pSmads. The abrogation of nuclear mechanosensation in Sun2 -/- fibroblasts disrupts force propagation into the nucleus, leading to a failure to produce pathogenic ECM but normal contractility (e.g. α-SMA expression) and protection from fibrosis.

Article Snippet: Sun2 -/- (strain B6;129S6-Sun2tm1Mhan/J) and C57BL/6 WT mice were obtained from Jackson Laboratories.

Techniques: Expressing

Journal: bioRxiv

Article Title: SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

doi: 10.64898/2026.03.15.711898

Figure Lengend Snippet: (A) CV-1 cells transfected with a scrambled (Scr) control siRNA or siRNA against SUN1 or SUN2 were harvested and the resulting whole cell extracts subjected to SDS-PAGE and immunoblotting with the indicated antibodies. β actin was used as a loading control. (B) CV-1 cells transfected with the indicated siRNA were infected with SV40, fixed, and stained for large T antigen (T-Ag). Data were normalized to the Scr control. (C) CV-1 cells transfected with the indicated siRNA were infected with SV40 and the resulting whole cell extracts were subjected to SDS-PAGE and immunoblotting. (D) The T-Ag band intensity in C was quantified by the FIJI software. Data were normalized to the Scr control. (E) Schematic of full-length (FL) SUN1 containing an N-terminal GFP tag (GFP-SUN1 FL), and a truncated SUN1 lacking the coiled-coil and SUN domain with also an N-terminal GFP tag (GFP-SUN1 ΔLU). (F) CV-1 cells transfected with the indicated constructs were fixed and stained for GFP (green) and counterstained with DAPI (blue). Scale bar: 10 µm. (G) CV-1 cells transfected with the Scr control siRNA or siRNA against SUN1 were also transfected with either the GFP-FLAG control construct or the indicated GFP-SUN1 construct. Cells were infected with SV40, fixed, and stained to assess T-Ag expression, as in 1B. Only GFP-expressing cells were analyzed and data were normalized to the Scr control with GFP-FLAG. (H) CV-1 cells were transfected with either Scr or siRNA against SUN1 or SUN2. The resulting whole cell extracts were subjected to SDS-PAGE and immunoblotted with the indicated antibodies. (I) The Nesprin-2 band intensity in H was quantified by the FIJI software and data were normalized to the Scr control. (J) CV-1 cells transfected with either Scr or siRNA against SUN1 or SUN2 were fixed and stained for Nesprin-2 (red) and counterstained with DAPI (blue). * p ≤ 0.05; ** p ≤ 0.01; ns= not significant. Scale bar: 10 µm

Article Snippet: Antibodies, along with the companies that were purchase from and the corresponding catalog numbers, are indicated: SUN1 (Invitrogen, MA5-47231), SUN2 (Proteintech, 27556-1-AP), β actin (Cell Signaling, 4967S), SV40 large T-antigen (Western blot: Santa Cruz Biotechnology, SC-53448; Immunofluorescence: Santa Cruz Biotechnology, SC-147), Nesprin-2 (IP and Immunofluorescence: Invitrogen, MA5-18075; Western blot: Bethyl, A305-393A), SV40 VP1 (Abcam, ab53977), SV40 VP2/3 (Abcam, ab53983), Bap31 (Invitrogen, MA 3002), Hsp90 (Santa Cruz Biotechnology, sc-13119), M2 FLAG (Millipore, F3165-1MG), FLAG (Millipore, F7425), BicD2 (Abcam, ab117818), KPNA1 (Proteintech, 18137-1-AP), KPNA2 (Invitrogen, 108191AP150UL), KPNA4 (Invitrogen, PA518239), Mab414 (Abcam, ab24609).

Techniques: Transfection, Control, SDS Page, Western Blot, Infection, Staining, Software, Construct, Expressing

Journal: bioRxiv

Article Title: SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

doi: 10.64898/2026.03.15.711898

Figure Lengend Snippet: (A) CV-1 cells transfected with either a scrambled (Scr) control siRNA or siRNA against SUN1 or SUN2 were infected with SV40 in the presence of actinomycin D (ActD). Cells were stained with anti-VP2/3 (green), mAb414 (red), and counterstained with DAPI (blue). Scale bar: 10 µm. (B) The percent of cells with a discrete VP2/3+ signal on or proximal to (i.e. within 0.3 µm) the nuclear membrane were quantified and normalized to the Scr control. (C) As in A, except stained with Bap31 (red). Scale bar: 10 µm. (D) CV-1 cells transfected with the indicated siRNA were infected with SV40 and processed using the ER-to-cytosol transport assay as described in (). The resulting cytosol and membrane fractions were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. ( E) The cytosol fraction from D was layered on a discontinuous sucrose gradient and centrifuged to generate individual fractions (see Materials and Methods). Fractions were subjected to SDS-PAGE followed by immunoblotting for VP1. The VP1 signal from fractions 1-7 (representing disassembled virus) and fraction 8 (representing assembled virus) was quantified and normalized to the Scr control. ** p ≤ 0.01; ns= not significant.

Article Snippet: Antibodies, along with the companies that were purchase from and the corresponding catalog numbers, are indicated: SUN1 (Invitrogen, MA5-47231), SUN2 (Proteintech, 27556-1-AP), β actin (Cell Signaling, 4967S), SV40 large T-antigen (Western blot: Santa Cruz Biotechnology, SC-53448; Immunofluorescence: Santa Cruz Biotechnology, SC-147), Nesprin-2 (IP and Immunofluorescence: Invitrogen, MA5-18075; Western blot: Bethyl, A305-393A), SV40 VP1 (Abcam, ab53977), SV40 VP2/3 (Abcam, ab53983), Bap31 (Invitrogen, MA 3002), Hsp90 (Santa Cruz Biotechnology, sc-13119), M2 FLAG (Millipore, F3165-1MG), FLAG (Millipore, F7425), BicD2 (Abcam, ab117818), KPNA1 (Proteintech, 18137-1-AP), KPNA2 (Invitrogen, 108191AP150UL), KPNA4 (Invitrogen, PA518239), Mab414 (Abcam, ab24609).

Techniques: Transfection, Control, Infection, Staining, Membrane, Transport Assay, SDS Page, Western Blot, Virus

Journal: bioRxiv

Article Title: SV40 exploits the Nesprin-2-SUN1-KPNA4 axis for stepwise targeting and entry into the host nucleus to promote infection

doi: 10.64898/2026.03.15.711898

Figure Lengend Snippet: (A) Schematic of full-length N-terminal FLAG-tagged SUN1 and SUN2, and of chimeric SUN proteins with swapped SUN domains. (B) CV-1 cells transfected with the indicated construct were fixed, stained for FLAG (green), and counterstained with DAPI (blue). Scale bar: 10 µm. (C) CV-1 cells transfected with the indicated construct and siRNA were infected with SV40, fixed, and stained for T-antigen, FLAG, and counterstained with DAPI. The percentage of transfected cells expressing T-antigen was quantified and normalized to the GFP+Scr control condition. (D) CV-1 cells transfected with the indicated construct were infected with SV40. FLAG-tagged proteins were IPed from the resulting whole cell extracts. The precipitated materials were subjected to PCR to detect SV40 genomic DNA or subjected to SDS-PAGE and immunoblotting. The dotted line indicates intervening lanes were removed with adjacent lanes spliced from the same immunoblot. ** p ≤ 0.01

Article Snippet: Antibodies, along with the companies that were purchase from and the corresponding catalog numbers, are indicated: SUN1 (Invitrogen, MA5-47231), SUN2 (Proteintech, 27556-1-AP), β actin (Cell Signaling, 4967S), SV40 large T-antigen (Western blot: Santa Cruz Biotechnology, SC-53448; Immunofluorescence: Santa Cruz Biotechnology, SC-147), Nesprin-2 (IP and Immunofluorescence: Invitrogen, MA5-18075; Western blot: Bethyl, A305-393A), SV40 VP1 (Abcam, ab53977), SV40 VP2/3 (Abcam, ab53983), Bap31 (Invitrogen, MA 3002), Hsp90 (Santa Cruz Biotechnology, sc-13119), M2 FLAG (Millipore, F3165-1MG), FLAG (Millipore, F7425), BicD2 (Abcam, ab117818), KPNA1 (Proteintech, 18137-1-AP), KPNA2 (Invitrogen, 108191AP150UL), KPNA4 (Invitrogen, PA518239), Mab414 (Abcam, ab24609).

Techniques: Transfection, Construct, Staining, Infection, Expressing, Control, SDS Page, Western Blot

Journal: Communications Biology

Article Title: Engineering CCR2/IFN-γ overexpression in enucleated mesenchymal stem cells enhances therapy for rheumatoid arthritis

doi: 10.1038/s42003-025-09248-5

Figure Lengend Snippet: A Schematic of workflow for genetic engineering and enucleation of hUC-MSCs. B Average diameter of MSCs or En-MSCs C/I . Mean ± s.e.m. (**** p < 0.0001). C Fluorescent confocal images of MSCs and En-MSCs C/I stained with F-actin for cytoskeleton, and Hochest for nucleus. Scale bar = 20 μm. Representative confocal image selected from three independent experiments. D Representative Electron microscopy images biologically independent samples of MSCs and En-MSCs C/I . Yellow arrow pointed to typical cell nucleus in MSC, Scale bar, 5 μm. E Flow cytometry analysis of DAPI staining post-enucleation. Compared to MSCs, En-MSCs C/I exhibited a DAPI-negative population of approximately 96.8%. F Quantification of En-MSCs C/I viability at 0, 24, 48, and 72 h post-enucleation using the Trypan Blue exclusion method. Data represent the percentage of viable (unstained) cells measured by automated cell counter. Mean ± s.e.m. ( n = 3, *** p < 0.0001). G Western Blot of LMNA/C, SUN2, Tublin, Vimentin, FAK, β-actin, and GAPDH in MSCs and En-MSCs C/I after 24 hours of adherence. The molecular weight marker (kDa) are shown. GAPDH served as the loading control. Lane order and sample identities are indicated on the blots. H Quantiffcation of the Western blot analysis. Data are represented as the Mean ± s.e.m. ( n = 6, *** p < 0.001). I Flow cytometry analysis of CCR2 and IFN-γ expression in MSCs, MSCs C/I , En-MSCs C/I at 24 and 48 h post-enucleation (from left to right). J Flow cytometry showing expression of negative cocktail, CD44, CD73, and CD90 at 24 and 48 h post-enucleation. All error bars denote Mean ± s.e.m.; dots indicate individual samples.

Article Snippet: The following primary antibodies were used: Tubulin (Abcam, ab18207,1:2000), LMNA/C (ABclonal, AD249,1:1000), SUN2 (ABclonal, A16325,1:1000), Vimentin (ABclonal, A11952,1:1000), FAK (ABclonal, A11195,1:1000), β-actin (Abcam, ab179467,1:1000), GAPDH (affinity, AF7021,1:1000).

Techniques: Staining, Electron Microscopy, Flow Cytometry, Western Blot, Molecular Weight, Marker, Control, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Cytomegalovirus disrupts Lamin A/C to control microtubule-mediated nuclear movement and cell migration

doi: 10.1073/pnas.2507831122

Figure Lengend Snippet: HCMV alters nuclear dynamics and cytoskeletal networks over the course of infection. ( A ) NHDFs were coinfected at MOI 1 with TB40/E-UL99-GFP and TB40/E-UL32-mCherry. Cells were imaged at 1 frame per 30 min between 67 and 138 h.p.i. Stills from Movie S1 are shown with the nucleus outlined, illustrating how its shape and movement changes during phases of rotation and the switch to cell migration. ( B ) The abundance of Lamin A/C, SUN2, and SUN1 across different cell types (MRC5; fibroblasts, ARPE19; epithelia) and viral strains (AD169; lab adapted, TB40/E; clinical) plotted from proteomics datasets in Hofstadter et al. ( 38 ). ( C – E ) NHDFs were mock infected or infected with TB40/E-UL99-mCherry at MOI 5 for the indicated times. ( C ) Samples were fixed and stained with antibodies against SUN2 (green) and gB (red), while DNA was stained with Hoechst (blue). White arrows point to migrating cells with characteristic oblong nuclei and an AC at the leading edge, which contain even lower levels of SUN2 than infected cells still at earlier stages of infection. An uninfected cell lacking an AC is also indicated with an orange arrow for reference. ( D ) Samples were fixed and stained with antibodies against β-actin (green) and gB (red), while DNA was stained with Hoechst (blue). White arrows highlight actin filament subsets that traverse the nucleus in uninfected cells. ( E ) Samples were fixed and stained with antibodies against acetylated tubulin (green) and gB (red), while DNA was stained with Hoechst (blue). Representative images are shown in A and C – E from multiple fields of view and at least three independent replicate experiments.

Article Snippet: The primary antibodies used for immunoblotting were Ac-K40 tubulin (Sigma-Aldrich: T6793), HCMV IE1/2 (Abcam: ab53495), SUN1 (Novus Biologicals: NBP1-87396), UL44 (Virusys: CA006); Lamin A/C (Proteintech: 10298-1-AP); Lamin B1 (Proteintech: 12987-1-AP); SUN2 (Novus Biologicals: NBP1-58289); gL [Hybridoma from the Chan Lab ( )], eIF4E (BD transduction laboratories: 610270), GFP (Cell Signaling Technologies: 2956S) and β-Actin (CST: 3700S).

Techniques: Infection, Migration, Staining

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Cytomegalovirus disrupts Lamin A/C to control microtubule-mediated nuclear movement and cell migration

doi: 10.1073/pnas.2507831122

Figure Lengend Snippet: HCMV pUL97 regulates infected cell motility independently of SUN2 and actin downregulation. ( A – D ) NHDFs were mock infected or infected with TB40/E-UL99-mCherry at MOI 5 and then treated with DMSO solvent control or 10 µM maribavir (MBV) for 6 d. ( A ) Samples were fixed and stained with antibodies against SUN2 (green) and gB (red), while DNA was stained with Hoechst (blue). ( B and C ) Samples were fixed and stained with antibodies against β-actin (green) and gB (red), while DNA was stained with Hoechst (blue). Insets in B show regions chosen for zooms in C . Representative images are shown for ( A – C ) derived from multiple independent fields of view and three or more independent experiments. ( D ) Graph plots the percentage of infected cells containing detectable actin filaments. n = 135 derived from three independent replicate experiments, two-tailed Student’s t test. **** P < 0.0001. Note that despite more actin filaments being detectable in MBV-treated cells, as shown in panels B and C these filaments (white arrows) were weak and difficult to detect compared with those of uninfected. ( E and F ) MBV-treatment suppresses infected cell migration. NHDFs expressing CLIP17-eGFP were infected with TB40/E-UL99-mCherry at MOI 2 and imaged at 1 frame per 30 min starting at 120 h.p.i. ( E ) Graph plots the displacement of the center of the nucleus above 30 µM under each condition. Bars represent mean ± SEM, ns = not significant, * P < 0. 05, one-way ANOVA test. n = 50 cells total (Mock-DMSO = 14, Mock-MBV = 11, HCMV-DMSO = 10, HCMV-MBV = 15) from 1 biological replicate; experiment was not repeated due to ability to assay multiple individual cells as well as the broader effects of pUL97 inhibition that potentially confound the interpretation of the overall phenotype. ( F ) Representative stills from Movie S2 , which shows the behavior of uninfected cells (white arrow) alongside infected cells at the late migration phase (red arrow) or newly infected in the early rotating phase (blue arrow) of infection.

Article Snippet: The primary antibodies used for immunoblotting were Ac-K40 tubulin (Sigma-Aldrich: T6793), HCMV IE1/2 (Abcam: ab53495), SUN1 (Novus Biologicals: NBP1-87396), UL44 (Virusys: CA006); Lamin A/C (Proteintech: 10298-1-AP); Lamin B1 (Proteintech: 12987-1-AP); SUN2 (Novus Biologicals: NBP1-58289); gL [Hybridoma from the Chan Lab ( )], eIF4E (BD transduction laboratories: 610270), GFP (Cell Signaling Technologies: 2956S) and β-Actin (CST: 3700S).

Techniques: Infection, Solvent, Control, Staining, Derivative Assay, Two Tailed Test, Migration, Expressing, Inhibition

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Cytomegalovirus disrupts Lamin A/C to control microtubule-mediated nuclear movement and cell migration

doi: 10.1073/pnas.2507831122

Figure Lengend Snippet: The effects of Lamin A/C mutants and SUN2 expression on actin filaments in HCMV-infected cells. ( A – C ) NHDFs stably expressing eGFP-tagged Lamin A/C variants; WT, nonphosphorylatable alanine mutant (S22A), or head and tail deletion mutant (ΔHC) were infected with TB40/E-UL99-mCherry at MOI 5 for 6 d. Cells were fixed in methanol (which quenches fluorescent proteins) and stained with antibodies against actin (green), gB (red), and GFP (turquoise), while DNA was stained with Hoechst (blue). ( A ) Images are representative of multiple fields of view derived from at least three independent experiments. ( B ) Enlarged examples of merged images from A which illustrate the partial restoration of actin filaments in some infected cells expressing the Lamin A/C S22A mutant; white arrows show examples of clear filaments while red arrows show examples of weak to no filament restoration. ( C ) Quantification of actin caps. n = 150 cells from three independent replicate experiments (wt: 1/50, 2/50, 1/50; S22A: 25/50, 20/50, 22/50, ΔHC: 0/50, 0/50, 1/50). Bars represent mean ± SEM; ns = not significant, **** P < 0.00001, one-way ANOVA test. ( D ) Lamin A/C expression does not restore SUN2 expression in infected cells. Lamin A/C-expressing cells were infected as described in A – C and then fixed and stained with antibodies against SUN2 (green), gB (red), and GFP (turquoise), while DNA was stained with Hoechst (blue). Representative images are shown from three independent replicate experiments. Note that methanol fixation was required for SUN2 imaging and incomplete quenching results in residual detection of concentrated GFP signal with the Lamin A/C-ΔHC variant, but its pattern is distinct and does not interfere with SUN2 detection. ( E and F ) SUN2 expression does not restore actin caps in infected cells. Control NHDFs or NHDFs expressing flag-tagged SUN2 were infected at MOI 5 for 6d then fixed and stained with antibodies against actin (green), gB (red), and Flag (turquoise), while DNA was stained with Hoechst (blue). ( E ) Representative images of uninfected or infected cells. ( F ) Quantification of actin caps in control or SUN2-expressing NHDFs infected with HCMV from three independent replicate experiments (control NHDFs: Mock; 55/35, 50/50, 44/44, or HCMV infected; 0/36, 0/44, 0/40; SUN2-expressing NHDFs: Mock; 47/47, 55/55, 48/48, or HCMV infected; 0/52, 0/47, 0/50). Bars represent mean ± SEM; **** P < 0.0001, two-way ANOVA test.

Article Snippet: The primary antibodies used for immunoblotting were Ac-K40 tubulin (Sigma-Aldrich: T6793), HCMV IE1/2 (Abcam: ab53495), SUN1 (Novus Biologicals: NBP1-87396), UL44 (Virusys: CA006); Lamin A/C (Proteintech: 10298-1-AP); Lamin B1 (Proteintech: 12987-1-AP); SUN2 (Novus Biologicals: NBP1-58289); gL [Hybridoma from the Chan Lab ( )], eIF4E (BD transduction laboratories: 610270), GFP (Cell Signaling Technologies: 2956S) and β-Actin (CST: 3700S).

Techniques: Expressing, Infection, Stable Transfection, Mutagenesis, Staining, Derivative Assay, Imaging, Variant Assay, Control

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Cytomegalovirus disrupts Lamin A/C to control microtubule-mediated nuclear movement and cell migration

doi: 10.1073/pnas.2507831122

Figure Lengend Snippet: Acetylated microtubules are required for HCMV-induced nuclear movement and cell migration. NHDFs were mock infected or infected with TB40/E-UL99-mCherry at MOI 5 and then treated with nontargeting control (Ctrl) siRNAs or either of two independent siRNAs targeting ATAT1 (ATAT1-A or B). ( A and B ) Samples were taken at 6 d.p.i. ( A ) Whole cell lysates were analyzed by Western blotting using the indicated antibodies. Blots are representative of three independent experiments. ( B ) Cells were fixed and stained with antibodies against SUN2 (green) or gB (red), while DNA was stained with Hoechst (blue). An uninfected cell or cell at the early stages of infection with no significant gB staining is indicated by the white arrow, adjacent to an infected cell where SUN2 levels are lower. Images are representative of phenotypes in multiple fields of view and derived from at least two independent experiments. ( C and D ) ATAT1 depletion reduces HCMV-induced cell migration. NHDFs expressing eGFP were infected at MOI 2 and treated with siRNAs as described in A and B . Time lapse imaging was performed at a frame rate of 1 image per 30 min starting at 120 h.p.i. ( C ) Graph plots the displacement of the center of the nucleus above 30 µM under each condition; bars represent mean ± SEM; n = 210 cells total, * P < 0.05, ns = not significant, one-way ANOVA test. ( D ) Representative stills from Movie S5 are shown with nuclei outlined and with arrows pointing to examples of cells that migrate in and out of the field of view in control siRNA-treated cells. By contrast, limited nuclear movement or cell migration is observed in ATAT1-depleted cells.

Article Snippet: The primary antibodies used for immunoblotting were Ac-K40 tubulin (Sigma-Aldrich: T6793), HCMV IE1/2 (Abcam: ab53495), SUN1 (Novus Biologicals: NBP1-87396), UL44 (Virusys: CA006); Lamin A/C (Proteintech: 10298-1-AP); Lamin B1 (Proteintech: 12987-1-AP); SUN2 (Novus Biologicals: NBP1-58289); gL [Hybridoma from the Chan Lab ( )], eIF4E (BD transduction laboratories: 610270), GFP (Cell Signaling Technologies: 2956S) and β-Actin (CST: 3700S).

Techniques: Migration, Infection, Control, Western Blot, Staining, Derivative Assay, Expressing, Imaging

Journal: The EMBO Journal

Article Title: Tension-sensitive LINC-RhoA signaling prevents chromatin bridge breakage in cytokinesis

doi: 10.1038/s44318-025-00565-3

Figure Lengend Snippet: ( A ) Actin patches intensity in control BE cells or cells treated with 1 μM SB273005 (integrin i) for 15 min. Mean ± SD from n cells from two independent experiments. Values in control were set to 1. P = 0.80 by Student’s t test. ( B ) Percentage of broken DNA bridges. Mean ± SD from four independent experiments ( n = 95, 53). P = 0.27 by Student’s t test. ( C – E ) Cartoons of cells exhibiting deformed nuclei. ( F – H ) DNA staining in cells with stretched or loose chromatin bridges, or without DNA bridges. ( I ) Nuclear chromatin shape in interphase cells without chromatin bridges, untransfected (control) cells with intact chromatin bridges, or cells transfected with Sun1/2 siRNA (siSun1/2), dominant-negative KASH (dnKASH) or Nesprin-2 siRNA (siNesprin-2) with intact chromatin bridges. Mean ± SD from n cells from two independent experiments. *** P = 4.04E-34 (interphase no bridges vs control), 7.89E-06 (interphase no bridges vs siSun1/2), 0.00037 (interphase no bridges vs dnKASH), 4.14E-13 (control vs siSun1/2), 2.46E-12 (control vs siNesprin-2) by ANOVA and Student’s t test. ( J ) Cartoon of the Sun1/2-Nesprin-2 LINC complex. SRs, spectrin repeats; INM/ONM, inner/outer nuclear membrane; CH, calponin homology. ( K – M ) Sun2 localization. ( N ) Percentage of cells exhibiting Sun2 nuclear lines. Cells were transfected with RhoA siRNA (siRhoA) or treated as in ( I ). Mean ± SD from three independent experiments ( n = 213, 79, 69, 72). *** P = 0.00098 (interphase no bridges vs control); ** P = 0.0011 (control vs dnKASH) by Student’s t test. ( O ) Nuclear chromatin shape in cells with intact or broken chromatin bridges. Mean ± SD from n cells from two independent experiments. *** P = 1.15E-06 (control intact vs siRhoA broken), 0.00032 (siRhoA intact vs siRhoA broken) by ANOVA and Student’s t test. ( P ) Cartoon showing the front and back of the nucleus where Nesprin-2, Sun1 or Sun2 intensity was measured. ( Q , R ) Nesprin-2 localization. ( S ) Front/back Nesprin-2 intensity. Mean ± SD from n cells from two independent experiments. *** P = 3.71E-21 (interphase no bridges vs control), 1.25E-10 (control vs siSun1/2), 1.91E-05 (control vs dnKASH) by ANOVA and Student’s t test. ( T ) Front/back Sun2 intensity. Mean ± SD from n cells from two independent experiments. *** P = 6.29E-18 by Student’s t test. ( U , V ) Nesprin-2 localization. Arrowheads show Sun2 nuclear lines or Nesprin-2 accumulation. ( W ) Actin patches. Intact arrows indicate actin patches and/or canal bases. Insets show high magnifications of the canal bases. Scale bars, 5 μm. ( X ) Percentage of cells exhibiting intact loose chromatin bridges. Mean ± SD from three independent experiments ( n = 188, 138, 113, 74). *** P = 7.29E-05 (control vs siSun1/2), 0.00025 (control vs siNesprin-2) by ANOVA and Student’s t test. ( Y ) Actin patches intensity. Mean ± SD from n cells from two independent experiments. Values in control were set to 1. Numbers below/next to each bar indicate n . *** P = 5.48E-20 (control vs siSun1/2), 1.004E-44 (control vs siNesprin-2) by ANOVA and Student’s t test. ( Z ) Percentage of cells exhibiting broken chromatin bridges. Mean ± SD from four independent experiments ( n = 95, 138, 138). *** P = 0.00013 (control vs siSun1/2), 0.00086 (control vs siNesprin-2) by ANOVA and Student’s t test. .

Article Snippet: Human RhoA (sc-29471; 5’-GGCAGAGAUAUGGCAAACA-3’), p115 RhoGEF (sc-41734; a pool of three individual siRNAs: 5’-CUGGAGGAGAUGCAACAUA-3’, 5’-CCAAGAGUGGAGACAAGAA-3’, 5’-CCGAUCACAAAGCCUUCUA-3’), PDZ RhoGEF (sc-45823; a pool of three individual siRNAs: 5’-GAACCUGCCUGAACUCAUA-3’, 5’-CAAGAGCCUGGAUCUUACA-3’, 5’-CCUCAGACAUGCAAGUGAA-3’), LARG RhoGEF (sc-41800; a pool of three individual siRNAs: 5’-GGAUGGAGCUGUAGUUACA-3’, 5’-CCAGAGCAUUGAAUUACUA-3’, 5’-CGAAGGAGAUAAUGAUGAA-3’), Sun1 (sc-106672; a pool of three individual siRNAs: 5’-GGAUGCCGUACAAGAAAGA-3’, 5’-GUAACUGCUGGGCAUUUAA-3’, 5’-CAAGGCACUUAAAGUGUUA-3’), Sun2 (sc-76612; a pool of three individual siRNAs: 5’-GGAAAUCCAGCAACAUGAA-3’, 5’-GACGUAUGGUGCUUGGUAU-3’, 5’-GCAUCAGCAAGACUCAGAA-3’), Nesprin-2 (Syne-2; sc-61630; a pool of three individual siRNAs: 5’-GGUAGAACGUCAACCUCAA-3’, 5’-CAAACAGCCUUCUCAUUAA-3’, 5’-GACUUCUGUUGUACUGAAA-3’), Lamin A/C (sc-35776, 5’-CUGGACUUCCAGAAGAACA-3’), PDZ-2 (5’-GAACCUGCCUGAACUCAUA-3’) and c-Src (sc-29228, 5’-GCAGUUGUAUGCUGUGGUU-3’) siRNAs were from Santa Cruz Biotechnology.

Techniques: Control, Staining, Transfection, Dominant Negative Mutation, Membrane

Journal: The EMBO Journal

Article Title: Tension-sensitive LINC-RhoA signaling prevents chromatin bridge breakage in cytokinesis

doi: 10.1038/s44318-025-00565-3

Figure Lengend Snippet: ( A , B ) RhoA localization in control BE cells or cells transfected with Sun1/2 siRNA (siSun1/2). Intact arrows indicate actin patches and/or canal bases. Broken arrows indicate broken DNA bridges. Scale bars, 5 μm. ( C ) RhoA intensity. Mean ± SD from n cells from two independent experiments. Values in control were set to 1. *** P = 1.01E-35 (control vs siSun1/2), 1.45E-38 (control vs siSun1/2 intact bridges) by ANOVA and Student’s t test. ( D ) Western blot analysis of total RhoA or α-tubulin (α-tub). ( E ) Actin patches intensity in GFP-positive cells. Cells were transfected with GFP:RhoA-G14V or GFP in the absence (control) or presence of siSun1/2. Mean ± SD from n cells from two independent experiments. Values in control GFP were set to 1. *** P = 2.83E-36 (control GFP vs GFP+siSun1/2), 1.79E-36 (GFP+siSun1/2 vs GFP:RhoA-G14V+siSun1/2) by ANOVA and Student’s t test. ( F ) Percentage of broken DNA bridges in GFP-positive cells. Mean ± SD from three independent experiments ( n = 125, 73, 83, 85). *** P = 0.00029; ** P = 0.0051 by ANOVA and Student’s t test. ( G ) Cartoons of Nesprin-2, mini-Nesprin-2 CB and CH* proteins. SRs spectrin repeats, ONM outer nuclear membrane, CH calponin homology. ( H ) Nuclear chromatin shape in cells expressing mini-Nesprin-2 CB or CH* proteins in the absence or presence of Nesprin-2 siRNA (siNesprin-2). Mean ± SD from n cells from two independent experiments. *** P = 1.01E-24 (interphase no bridges vs control GFP), 1.33E-07 (interphase no bridges vs GFP+siNesprin-2), 3.46E-06 (interphase no bridges vs CH*+siNesprin-2), 2.63E-06 (control GFP vs GFP+siNesprin-2), 5.98E-05 (GFP+siNesprin-2 vs CB+siNesprin-2), 5.92E-05 (CB+siNesprin-2 vs CH*+siNesprin-2) by ANOVA and Student’s t test. ( I ) Percentage of cells exhibiting Sun2 nuclear lines. Mean ± SD from three independent experiments ( n = 215, 64, 52, 65). ** P = 0.0062 (interphase no bridges vs CH*+siNesprin-2), 0.0018 (CB+siNesprin-2 vs CH*+siNesprin-2) by ANOVA and Student’s t test. ( J ) Percentage of intact loose DNA bridges in GFP- or YFP-positive cells. Mean ± SD from three independent experiments ( n = 143, 70, 102, 107). *** P = 0.00016 (control GFP vs GFP+siNesprin-2), 1.27E-05 (CB+siNesprin-2 vs CH*+siNesprin-2); ** P = 0.0035 by ANOVA and Student’s t test. ( K ) Chromatin bridges and actin patches in cells expressing mini-Nesprin-2 CB. ( L ) Actin patches intensity in GFP- or YFP-positive cells. Values in control GFP were set to 1. Mean ± SD from n cells from two independent experiments. *** P = 4.97E-88 (control GFP vs GFP+siNesprin-2), 1.05E-83 (control GFP vs CB+siNesprin-2), 1.63E-93 (control GFP vs CH*+siNesprin-2) by ANOVA and Student’s t test. ( M ) Percentage of broken DNA bridges in GFP- or YFP-positive cells. Mean ± SD from four independent experiments ( n = 125, 92, 84, 107). *** P = 3.83E-06 (control GFP vs GFP+siNesprin-2), 1.34E-06 (control GFP vs CB+siNesprin-2), 0.00072 (control GFP vs CH*+siNesprin-2); ** P = 0.009 (GFP+siNesprin-2 vs CB+siNesprin-2), 0.0079 (CB+siNesprin-2vs CH*+siNesprin-2) by ANOVA and Student’s t test. ( N , O ) Chromatin bridges and actin patches in cells expressing mini-Nesprin-2 CH* protein. Intact arrows indicate actin patches and/or canal bases. Broken arrows indicate broken DNA bridges. Insets show high magnifications of the canal bases. Scale bars, 5 μm. ( P ) Nuclear chromatin shape in cells transfected with Lamin A/C siRNA (siLamin A/C), or as in ( A , B ). Mean ± SD from n cells from two independent experiments. *** P = 1.28E-05 (interphase no bridges vs siLaminA/C), 4.44E-12 (control vs siLaminA/C) by ANOVA and Student’s t test. ( Q ) Percentage of intact loose DNA bridges. Mean ± SD from three independent experiments ( n = 188, 77). *** P = 0.00021 by ANOVA and Student’s t test. ( R ) Front/back Nesprin-2 intensity. Mean ± SD from n cells from two independent experiments. *** P = 4.34E-21 (interphase no bridges vs siRhoA), 1.24E-15 (control vs siLaminA/C) by ANOVA and Student’s t test. ( S ) Actin patches intensity. Mean ± SD from n cells from two independent experiments. Values in control were set to 1. Numbers below/next to each bar indicate n . *** P = 8.75E-56 by Student’s t test. .

Article Snippet: Human RhoA (sc-29471; 5’-GGCAGAGAUAUGGCAAACA-3’), p115 RhoGEF (sc-41734; a pool of three individual siRNAs: 5’-CUGGAGGAGAUGCAACAUA-3’, 5’-CCAAGAGUGGAGACAAGAA-3’, 5’-CCGAUCACAAAGCCUUCUA-3’), PDZ RhoGEF (sc-45823; a pool of three individual siRNAs: 5’-GAACCUGCCUGAACUCAUA-3’, 5’-CAAGAGCCUGGAUCUUACA-3’, 5’-CCUCAGACAUGCAAGUGAA-3’), LARG RhoGEF (sc-41800; a pool of three individual siRNAs: 5’-GGAUGGAGCUGUAGUUACA-3’, 5’-CCAGAGCAUUGAAUUACUA-3’, 5’-CGAAGGAGAUAAUGAUGAA-3’), Sun1 (sc-106672; a pool of three individual siRNAs: 5’-GGAUGCCGUACAAGAAAGA-3’, 5’-GUAACUGCUGGGCAUUUAA-3’, 5’-CAAGGCACUUAAAGUGUUA-3’), Sun2 (sc-76612; a pool of three individual siRNAs: 5’-GGAAAUCCAGCAACAUGAA-3’, 5’-GACGUAUGGUGCUUGGUAU-3’, 5’-GCAUCAGCAAGACUCAGAA-3’), Nesprin-2 (Syne-2; sc-61630; a pool of three individual siRNAs: 5’-GGUAGAACGUCAACCUCAA-3’, 5’-CAAACAGCCUUCUCAUUAA-3’, 5’-GACUUCUGUUGUACUGAAA-3’), Lamin A/C (sc-35776, 5’-CUGGACUUCCAGAAGAACA-3’), PDZ-2 (5’-GAACCUGCCUGAACUCAUA-3’) and c-Src (sc-29228, 5’-GCAGUUGUAUGCUGUGGUU-3’) siRNAs were from Santa Cruz Biotechnology.

Techniques: Control, Transfection, Western Blot, Membrane, Expressing