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: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting KMT5C Suppresses Lung Cancer Progression and Enhances the Efficacy of Immunotherapy.

doi: 10.1002/advs.202407575

Figure Lengend Snippet: Figure 1. Upregulation of KMT5C in NSCLC is associated with cancer progression and poor prognosis. A–C) Comparison the mRNA level of KMT5C between normal and tumor tissues in the TCGA_LUAD (A), TCGA_LUSC (B) and TCGA_NSCLC (C) cohorts. D) Real-time PCR analysis the expression level of KMT5C in NSCLC tumor tissues (n = 17) and paired non-tumor tissues (n = 17). E) Western blot analysis and statistical the level of KMT5C in NSCLC tumor tissues (T, n = 7) and paired non-tumor tissues (N, n = 7). F,G) Representative IHC staining images (F) and statistical data (G) of KMT5C level in NSCLC tumor tissues (n = 173) and peritumor tissues (n = 173). Scale bar, 50 μm. H) Relative IHC level of KMT5C between tumor different

Article Snippet: Antibodies: Antibodies for western blot included: KMT5C (A16325, ABclonal, 1:1000), STING (19851-1-AP, Proteintech, 1:2000), p-STING (n = 5).

Techniques: Comparison, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Immunohistochemistry

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting KMT5C Suppresses Lung Cancer Progression and Enhances the Efficacy of Immunotherapy.

doi: 10.1002/advs.202407575

Figure Lengend Snippet: Figure 2. KMT5C promotes NSCLC cell proliferation, migration and invasion in vitro. A) Western blot analysis the A549 cells infected with the lentivirus expressing KMT5C knockdown shRNA (shKMT5C-1 and shKMT5C-1) or control shRNA (shNC). B) CCK8 assays analysis the cell proliferation from A549 cells infected with the indicated lentivirus (n = 6). C) CCK8 assays analysis the cell proliferation from A549 cells infected with control and KMT5C overexpression lentivirus (n = 6). D) Representative images and the statistical data of colony formation from the A549 cells infected with the indicated lentivirus (n = 3). E) Cell cycle analysis of A549 cells after stable knockdown of KMT5C (n = 4). F) Cell cycle analysis of A549 cells after KMT5C overex-

Article Snippet: Antibodies: Antibodies for western blot included: KMT5C (A16325, ABclonal, 1:1000), STING (19851-1-AP, Proteintech, 1:2000), p-STING (n = 5).

Techniques: Migration, In Vitro, Western Blot, Infection, Expressing, Knockdown, shRNA, Control, Over Expression, Cell Cycle Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting KMT5C Suppresses Lung Cancer Progression and Enhances the Efficacy of Immunotherapy.

doi: 10.1002/advs.202407575

Figure Lengend Snippet: Figure 3. Knockdown of KMT5C in NSCLC suppresses tumor growth and metastasis in mice. A–C) A549 cells stably expressing the KMT5C knockdown shRNA (shKMT5C-1 and shKMT5C-2) and control shRNA (shNC) were subcutaneously injected in nude mice, respectively. Shown are average tumor volumes over time (A) and representative image (B) and weight (C) of tumors as the indicated (n = 6). Scale bar, 1 cm. D, E) Representative images of HE and IHC Ki-67 staining (D), and the IHC scores of Ki-67 expression (E) in the above tissues as indicated (n = 6). Scale bar, 50 μm. F,G) LLC cells stably expressing the Kmt5c knockdown shRNA (shKmt5c-1 and shKmt5c-2) and control shRNA (Control) were subcutaneously injected in C57BL/6

Article Snippet: Antibodies: Antibodies for western blot included: KMT5C (A16325, ABclonal, 1:1000), STING (19851-1-AP, Proteintech, 1:2000), p-STING (n = 5).

Techniques: Knockdown, Stable Transfection, Expressing, shRNA, Control, Injection, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting KMT5C Suppresses Lung Cancer Progression and Enhances the Efficacy of Immunotherapy.

doi: 10.1002/advs.202407575

Figure Lengend Snippet: Figure 4. KMT5C knockdown impairs DNA damage response to activate the STING-IRF3, downstream type I IFN signaling and induces CCL5 expression. A,B) GSEA analysis the RNA-seq data from A549 cells with KMT5C knockdown (shKMT5C) or control (shNC) (n = 3). Shown are the images of top upregulated pathways (A) (shKMT5C VS shNC) and top downregulated pathways (B) (shKMT5C VS shNC). C Heatmap demonstration of the gene expression levels of RAD51, RAD52, BRCA1, XRCC2 and XRCC3 in the RNA-seq data from A549 cells after stably KMT5C knockdown as indicated. D) Real-time PCR analysis the relative mRNA levels in A549 cells as indicated (n = 3). E) Representative images of IHC 𝛾H2AX staining in tumor tissues

Article Snippet: Antibodies: Antibodies for western blot included: KMT5C (A16325, ABclonal, 1:1000), STING (19851-1-AP, Proteintech, 1:2000), p-STING (n = 5).

Techniques: Knockdown, Expressing, RNA Sequencing, Control, Gene Expression, Stable Transfection, Real-time Polymerase Chain Reaction, Staining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting KMT5C Suppresses Lung Cancer Progression and Enhances the Efficacy of Immunotherapy.

doi: 10.1002/advs.202407575

Figure Lengend Snippet: Figure 5. CCL5 is required for KMT5C knockdown-mediated the improvement of tumor immune microenvironment. A–C) LLC cells with Control or Kmt5c knockdown were subcutaneously injected in C57BL/6 mice and nude mice. Shown are the representative image of tumors (A), and statistical data of tumor volume (B) and weight (C) as the indicated (n = 8). Scale bar, 1 cm. D–F) LLC cells stably expressing the indicated shRNA were subcutaneously injected in C57BL/6 mice, respectively. Shown are the representative image of tumors (D), and statistical data of tumor volume (E) and weight (F) as the indicated (n = 7). Scale bar, 1 cm. G) Relative proportions of CD45+, CD3+, CD4+ and CD8+ T cells in tumor tissues were analyzed by flow cytometry

Article Snippet: Antibodies: Antibodies for western blot included: KMT5C (A16325, ABclonal, 1:1000), STING (19851-1-AP, Proteintech, 1:2000), p-STING (n = 5).

Techniques: Knockdown, Control, Injection, Stable Transfection, Expressing, shRNA, Cytometry

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting KMT5C Suppresses Lung Cancer Progression and Enhances the Efficacy of Immunotherapy.

doi: 10.1002/advs.202407575

Figure Lengend Snippet: Figure 6. KMT5C inhibitor potentiates the response of NSCLC to anti-PD-1 therapy. A–C) LLC cells were subcutaneously injected in C57BL/6 mice (n = 8 mice/group). After 3–5 days post-injection, mice were treated with vehicle control and A196 (20 mg per kg daily, intragastrically (i.g)), anti-PD-1 antibody (100 μg per time, every 2 days for a total of 4 intraperitoneal injection (i.p)) or a combined respectively until the end of experiments. Shown are the representative image of tumors (A), and statistical data of tumor volume (B) and weight (C) as the indicated (n = 8). Scale bar, 1 cm. D) Relative proportions of CD45+, CD3+, CD4+ and CD8+ T cells in tumor tissues were analyzed by flow cytometry (n = 5). E) Gating strategies for analysis of

Article Snippet: Antibodies: Antibodies for western blot included: KMT5C (A16325, ABclonal, 1:1000), STING (19851-1-AP, Proteintech, 1:2000), p-STING (n = 5).

Techniques: Injection, Control, Cytometry

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Targeting KMT5C Suppresses Lung Cancer Progression and Enhances the Efficacy of Immunotherapy.

doi: 10.1002/advs.202407575

Figure Lengend Snippet: Figure 7. KMT5C high expression in NSCLC is associated with immunotherapy resistance and worse prognosis to ICB therapy. A,B) Correlation analysis of the cytotoxic T lymphocyte (CTL) level and the expression of KMT5C in lung cancer (GSE30219) and liver cancer (GSE54236) (A), and two melanoma ICB therapy cohorts (Gide2019_PD1+CTLA4 and VanAllen2015_CTLA4) (B) from the TIDE database (http://tide.dfci.harvard.edu/). The Pearson coeffi- cient was used to evaluate correlations. C) Kaplan-Meier analysis of the association between CTL levels with overall survival (OS) in lung cancer patients with KMT5C high or low expression group from the TIDE database (http://tide.dfci.harvard.edu/). D) The expression level of KMT5C in pancancer_anti-

Article Snippet: Antibodies: Antibodies for western blot included: KMT5C (A16325, ABclonal, 1:1000), STING (19851-1-AP, Proteintech, 1:2000), p-STING (n = 5).

Techniques: Expressing

Journal: bioRxiv

Article Title: Dissecting Force Transmission across SUN Proteins Using Nuclear Tension Sensors

doi: 10.1101/2025.02.17.638756

Figure Lengend Snippet: ( A ) Scheme of SUN proteins, three designs of the SUN-based nuclear tension sensor (NuTS), and truncated tail-less (TL). ( B ) The working mechanism of the mechanical sensing of NuTS. FRET ratio is negatively correlated with mechanical force. Low FRET of NuTS represents large force transmission on SUN protein, vice versa. ( C ) Representative FRET ratiometric images of TL1 and three designs of NuTS1 transiently expressed in HeLa-WT, C2C12-WT, and C2C12- SUN1 KO cells, respectively. Scale bars, 20 μm for large view, and 10 μm for zoom in. ( D ) The quantified FRET ratio (Mean ± SEM) of TL1 and series NuTS1 from ( C ). n = 81, 76, 101, 81 (HeLa), n = 59, 62, 93, 85 (C2C12-WT), and n = 82, 65, 99, 119 (C2C12- SUN1 KO). ( E ) Fold changes of three designs of NuTS1 calculated by (TL1-NuTS1)/TL1*100% from ( D ). KL-NuTS1 showed the most FRET ratio change compared to TL1 in three types of cells. ( F ) Representative FRET ratiometric images of TL2 and three designs of NuTS2 transiently expressed in HeLa-WT, C2C12-WT, and C2C12- SUN2 KO cells, respectively. Scale bars, 20 μm for large view, and 10 μm for zoom in. ( G ) The quantified FRET ratio (Mean ± SEM) of TL2 and series NuTS2 from ( F ). n = 114, 48, 116, 104 (HeLa), n = 103, 54, 105, 104 (C2C12-WT), and n = 88, 54, 102, 101 (C2C12- SUN2 KO). ( H ) Fold changes of three designs of NuTS2 calculated by (TL2-NuTS2)/TL2*100% from ( G ). KL-NuTS2 and CSD-NuTS2 displayed higher FRET ratio changes than SD-NuTS2 in all type of cells. Three biological replicates were performed for all experiments, respectively. Ordinary one-way ANOVA Tukey’s multiple comparisons.

Article Snippet: The primary antibody, Anti-SUN1 antibody (1:200; abcam, cat #ab103021) for SUN1, Anti-SUN2 antibody (1:500; abcam, cat #ab124916) for SUN2, Syne-2 Antibody (F-11) (1:200; Santa Cruz, cat #sc-398616) for nesprin 2, lamin A/C (4C11) Mouse mAb (1:200; Cell Signaling Technology, cat #4777S) for lamin A/C, respectively, was overnight incubation at 4°C and rinsed with PBST (PBS with 0.1% Tween® 20).

Techniques: Transmission Assay

Journal: bioRxiv

Article Title: Dissecting Force Transmission across SUN Proteins Using Nuclear Tension Sensors

doi: 10.1101/2025.02.17.638756

Figure Lengend Snippet: The sequence information was from National Library of Medicine, National Center for Biotechnology Information (Gene ID: 77053 for Sun1 Sad1 and UNC84 domain containing 2 [ Mus musculus (house mouse)], and 223697 for Sun2 Sad1 and UNC84 domain containing 2 [ Mus musculus (house mouse)]). The subdomain and amino acids sequence were from UniProt (Protein ID: Q9D666 for SUN domain-containing protein 1 and Q8BJS4 for SUN domain-containing protein 2). ( A ) for SUN1 and ( B ) for SUN2.

Article Snippet: The primary antibody, Anti-SUN1 antibody (1:200; abcam, cat #ab103021) for SUN1, Anti-SUN2 antibody (1:500; abcam, cat #ab124916) for SUN2, Syne-2 Antibody (F-11) (1:200; Santa Cruz, cat #sc-398616) for nesprin 2, lamin A/C (4C11) Mouse mAb (1:200; Cell Signaling Technology, cat #4777S) for lamin A/C, respectively, was overnight incubation at 4°C and rinsed with PBST (PBS with 0.1% Tween® 20).

Techniques: Sequencing

Journal: bioRxiv

Article Title: Dissecting Force Transmission across SUN Proteins Using Nuclear Tension Sensors

doi: 10.1101/2025.02.17.638756

Figure Lengend Snippet: ( A ) Representative immunostaining of SUN1 in SUN1 KO and WT C2C12. Green for SUN1, red for F-actin, and blue for DAPI. Scale bars, 20 μm. ( B ) Representative immunostaining of SUN2 in SUN2 KO and WT C2C12. Green for SUN2, red for F-actin, and blue for DAPI. Scale bars, 20 μm.

Article Snippet: The primary antibody, Anti-SUN1 antibody (1:200; abcam, cat #ab103021) for SUN1, Anti-SUN2 antibody (1:500; abcam, cat #ab124916) for SUN2, Syne-2 Antibody (F-11) (1:200; Santa Cruz, cat #sc-398616) for nesprin 2, lamin A/C (4C11) Mouse mAb (1:200; Cell Signaling Technology, cat #4777S) for lamin A/C, respectively, was overnight incubation at 4°C and rinsed with PBST (PBS with 0.1% Tween® 20).

Techniques: Immunostaining

Journal: bioRxiv

Article Title: Dissecting Force Transmission across SUN Proteins Using Nuclear Tension Sensors

doi: 10.1101/2025.02.17.638756

Figure Lengend Snippet: ( A ) Scheme of SUN proteins, three designs of the SUN-based nuclear tension sensor (NuTS), and truncated tail-less (TL). ( B ) The working mechanism of the mechanical sensing of NuTS. FRET ratio is negatively correlated with mechanical force. Low FRET of NuTS represents large force transmission on SUN protein, vice versa. ( C ) Representative FRET ratiometric images of TL1 and three designs of NuTS1 transiently expressed in HeLa-WT, C2C12-WT, and C2C12- SUN1 KO cells, respectively. Scale bars, 20 μm for large view, and 10 μm for zoom in. ( D ) The quantified FRET ratio (Mean ± SEM) of TL1 and series NuTS1 from ( C ). n = 81, 76, 101, 81 (HeLa), n = 59, 62, 93, 85 (C2C12-WT), and n = 82, 65, 99, 119 (C2C12- SUN1 KO). ( E ) Fold changes of three designs of NuTS1 calculated by (TL1-NuTS1)/TL1*100% from ( D ). KL-NuTS1 showed the most FRET ratio change compared to TL1 in three types of cells. ( F ) Representative FRET ratiometric images of TL2 and three designs of NuTS2 transiently expressed in HeLa-WT, C2C12-WT, and C2C12- SUN2 KO cells, respectively. Scale bars, 20 μm for large view, and 10 μm for zoom in. ( G ) The quantified FRET ratio (Mean ± SEM) of TL2 and series NuTS2 from ( F ). n = 114, 48, 116, 104 (HeLa), n = 103, 54, 105, 104 (C2C12-WT), and n = 88, 54, 102, 101 (C2C12- SUN2 KO). ( H ) Fold changes of three designs of NuTS2 calculated by (TL2-NuTS2)/TL2*100% from ( G ). KL-NuTS2 and CSD-NuTS2 displayed higher FRET ratio changes than SD-NuTS2 in all type of cells. Three biological replicates were performed for all experiments, respectively. Ordinary one-way ANOVA Tukey’s multiple comparisons.

Article Snippet: The primary antibody, Anti-SUN1 antibody (1:200; abcam, cat #ab103021) for SUN1, Anti-SUN2 antibody (1:500; abcam, cat #ab124916) for SUN2, Syne-2 Antibody (F-11) (1:200; Santa Cruz, cat #sc-398616) for nesprin 2, lamin A/C (4C11) Mouse mAb (1:200; Cell Signaling Technology, cat #4777S) for lamin A/C, respectively, was overnight incubation at 4°C and rinsed with PBST (PBS with 0.1% Tween® 20).

Techniques: Transmission Assay

Journal: bioRxiv

Article Title: Dissecting Force Transmission across SUN Proteins Using Nuclear Tension Sensors

doi: 10.1101/2025.02.17.638756

Figure Lengend Snippet: The sequence information was from National Library of Medicine, National Center for Biotechnology Information (Gene ID: 77053 for Sun1 Sad1 and UNC84 domain containing 2 [ Mus musculus (house mouse)], and 223697 for Sun2 Sad1 and UNC84 domain containing 2 [ Mus musculus (house mouse)]). The subdomain and amino acids sequence were from UniProt (Protein ID: Q9D666 for SUN domain-containing protein 1 and Q8BJS4 for SUN domain-containing protein 2). ( A ) for SUN1 and ( B ) for SUN2.

Article Snippet: The primary antibody, Anti-SUN1 antibody (1:200; abcam, cat #ab103021) for SUN1, Anti-SUN2 antibody (1:500; abcam, cat #ab124916) for SUN2, Syne-2 Antibody (F-11) (1:200; Santa Cruz, cat #sc-398616) for nesprin 2, lamin A/C (4C11) Mouse mAb (1:200; Cell Signaling Technology, cat #4777S) for lamin A/C, respectively, was overnight incubation at 4°C and rinsed with PBST (PBS with 0.1% Tween® 20).

Techniques: Sequencing

Journal: bioRxiv

Article Title: Dissecting Force Transmission across SUN Proteins Using Nuclear Tension Sensors

doi: 10.1101/2025.02.17.638756

Figure Lengend Snippet: ( A ) Representative immunostaining of SUN1 in SUN1 KO and WT C2C12. Green for SUN1, red for F-actin, and blue for DAPI. Scale bars, 20 μm. ( B ) Representative immunostaining of SUN2 in SUN2 KO and WT C2C12. Green for SUN2, red for F-actin, and blue for DAPI. Scale bars, 20 μm.

Article Snippet: The primary antibody, Anti-SUN1 antibody (1:200; abcam, cat #ab103021) for SUN1, Anti-SUN2 antibody (1:500; abcam, cat #ab124916) for SUN2, Syne-2 Antibody (F-11) (1:200; Santa Cruz, cat #sc-398616) for nesprin 2, lamin A/C (4C11) Mouse mAb (1:200; Cell Signaling Technology, cat #4777S) for lamin A/C, respectively, was overnight incubation at 4°C and rinsed with PBST (PBS with 0.1% Tween® 20).

Techniques: Immunostaining

Journal: bioRxiv

Article Title: Dissecting Force Transmission across SUN Proteins Using Nuclear Tension Sensors

doi: 10.1101/2025.02.17.638756

Figure Lengend Snippet: ( A ) Scheme of SUN proteins, three designs of the SUN-based nuclear tension sensor (NuTS), and truncated tail-less (TL). ( B ) The working mechanism of the mechanical sensing of NuTS. FRET ratio is negatively correlated with mechanical force. Low FRET of NuTS represents large force transmission on SUN protein, vice versa. ( C ) Representative FRET ratiometric images of TL1 and three designs of NuTS1 transiently expressed in HeLa-WT, C2C12-WT, and C2C12- SUN1 KO cells, respectively. Scale bars, 20 μm for large view, and 10 μm for zoom in. ( D ) The quantified FRET ratio (Mean ± SEM) of TL1 and series NuTS1 from ( C ). n = 81, 76, 101, 81 (HeLa), n = 59, 62, 93, 85 (C2C12-WT), and n = 82, 65, 99, 119 (C2C12- SUN1 KO). ( E ) Fold changes of three designs of NuTS1 calculated by (TL1-NuTS1)/TL1*100% from ( D ). KL-NuTS1 showed the most FRET ratio change compared to TL1 in three types of cells. ( F ) Representative FRET ratiometric images of TL2 and three designs of NuTS2 transiently expressed in HeLa-WT, C2C12-WT, and C2C12- SUN2 KO cells, respectively. Scale bars, 20 μm for large view, and 10 μm for zoom in. ( G ) The quantified FRET ratio (Mean ± SEM) of TL2 and series NuTS2 from ( F ). n = 114, 48, 116, 104 (HeLa), n = 103, 54, 105, 104 (C2C12-WT), and n = 88, 54, 102, 101 (C2C12- SUN2 KO). ( H ) Fold changes of three designs of NuTS2 calculated by (TL2-NuTS2)/TL2*100% from ( G ). KL-NuTS2 and CSD-NuTS2 displayed higher FRET ratio changes than SD-NuTS2 in all type of cells. Three biological replicates were performed for all experiments, respectively. Ordinary one-way ANOVA Tukey’s multiple comparisons.

Article Snippet: The primary antibody, Anti-SUN1 antibody (1:200; abcam, cat #ab103021) for SUN1, Anti-SUN2 antibody (1:500; abcam, cat #ab124916) for SUN2, Syne-2 Antibody (F-11) (1:200; Santa Cruz, cat #sc-398616) for nesprin 2, lamin A/C (4C11) Mouse mAb (1:200; Cell Signaling Technology, cat #4777S) for lamin A/C, respectively, was overnight incubation at 4°C and rinsed with PBST (PBS with 0.1% Tween® 20).

Techniques: Transmission Assay

Journal: bioRxiv

Article Title: Dissecting Force Transmission across SUN Proteins Using Nuclear Tension Sensors

doi: 10.1101/2025.02.17.638756

Figure Lengend Snippet: The sequence information was from National Library of Medicine, National Center for Biotechnology Information (Gene ID: 77053 for Sun1 Sad1 and UNC84 domain containing 2 [ Mus musculus (house mouse)], and 223697 for Sun2 Sad1 and UNC84 domain containing 2 [ Mus musculus (house mouse)]). The subdomain and amino acids sequence were from UniProt (Protein ID: Q9D666 for SUN domain-containing protein 1 and Q8BJS4 for SUN domain-containing protein 2). ( A ) for SUN1 and ( B ) for SUN2.

Article Snippet: The primary antibody, Anti-SUN1 antibody (1:200; abcam, cat #ab103021) for SUN1, Anti-SUN2 antibody (1:500; abcam, cat #ab124916) for SUN2, Syne-2 Antibody (F-11) (1:200; Santa Cruz, cat #sc-398616) for nesprin 2, lamin A/C (4C11) Mouse mAb (1:200; Cell Signaling Technology, cat #4777S) for lamin A/C, respectively, was overnight incubation at 4°C and rinsed with PBST (PBS with 0.1% Tween® 20).

Techniques: Sequencing

Journal: bioRxiv

Article Title: Dissecting Force Transmission across SUN Proteins Using Nuclear Tension Sensors

doi: 10.1101/2025.02.17.638756

Figure Lengend Snippet: ( A ) Representative immunostaining of SUN1 in SUN1 KO and WT C2C12. Green for SUN1, red for F-actin, and blue for DAPI. Scale bars, 20 μm. ( B ) Representative immunostaining of SUN2 in SUN2 KO and WT C2C12. Green for SUN2, red for F-actin, and blue for DAPI. Scale bars, 20 μm.

Article Snippet: The primary antibody, Anti-SUN1 antibody (1:200; abcam, cat #ab103021) for SUN1, Anti-SUN2 antibody (1:500; abcam, cat #ab124916) for SUN2, Syne-2 Antibody (F-11) (1:200; Santa Cruz, cat #sc-398616) for nesprin 2, lamin A/C (4C11) Mouse mAb (1:200; Cell Signaling Technology, cat #4777S) for lamin A/C, respectively, was overnight incubation at 4°C and rinsed with PBST (PBS with 0.1% Tween® 20).

Techniques: Immunostaining