Journal: Scientific reports

Article Title: Emerin deficiency drives MCF7 cells to an invasive phenotype.

doi: 10.1038/s41598-024-70752-5

Figure Lengend Snippet: Fig. 9. Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451). Nuclei are blue, emerin is brown, and arrows denote emerin staining in certain images for reference. As severity of cases increases, there is a visible reduction in emerin expression at the nuclear envelope and more deformed nuclei are present. (B) Quantification of emerin staining on IHC-stained patient samples using 0–3, with 0 having no staining at the nuclear periphery and 3 having complete, dark rim staining. N = 159 total samples, *P < 0.05 compared to normal tissue, one-way ANOVA and Dunnett’s test. Error bars represent standard deviation. (C) Representative tissue microarray staining of emerin in 183 patients using emerin monoclonal antibodies (Leica, NCL-Emerin) or secondary alone (Vector Lab, cat#: MP-7452) using the same samples used in A. Nuclei are blue and emerin is brown. As aggressiveness of cases increases, there is a visible reduction in emerin expression and more deformed nuclei are present. (D) Quantification of emerin staining using the 0 to 3 grading system. N = 183 total samples #P < 0.02 compared to all non-cancerous tissue, *P < 0.0062 compared to both normal and benign tissue, one-way ANOVA and Dunnett’s test. Error bars represent standard deviation.

Article Snippet: Reduced emerin expression at the nuclear periphery correlates with breast cancer invasiveness in patients. (A) Representative tissue microarray staining of emerin in 159 patients using emerin polyclonal antibodies (Proteintech, cat# 10351-1-AP) or secondary alone (Vector Lab, cat#: MP-7451).

Techniques: Expressing, Microarray, Staining, Plasmid Preparation, Standard Deviation, Bioprocessing

Journal: EMBO Reports

Article Title: SUN2 mediates calcium-triggered nuclear actin polymerization to cluster active RNA polymerase II

doi: 10.1038/s44319-024-00274-8

Figure Lengend Snippet: ( A ) Representative spinning disc confocal slices of a NIH3T3 cell stably expressing nAC-tagGFP showing rapid nuclear F-actin assembly upon addition of 1 µM A23187. Scale bar = 5 µm. (min:sec after drug treatment). ( B ) Model of the nuclear envelope (NE) spanning linker of nucleoskeleton and cytoskeleton (LINC) complex. It consists of SUN proteins (red) located in the inner nuclear membrane (INM) which bind to the KASH domain of nesprin proteins (orange) residing in the outer nuclear membrane (ONM). In the cytosol, nesprins bind to components of the cytoskeleton including F-actin. Inside the nuclear compartment, SUN proteins interact with nuclear lamins and other inner nuclear membrane proteins like emerin (green). The integrity and mechanotransduction function of the LINC complex can be disrupted by introducing dominant-negative KASH (dnKASH) domains which displace endogenous nesprins from the NE. ( C ) NIH3T3 cells stably expressing nAC-tagGFP that were transfected with non-targeting control siRNA (siCtrl, n = 611) or siRNA targeting emerin (siEmd, n = 528) were stimulated with 1 μM A23187 and scored for the occurrence of nuclear actin assembly. Average percentage of positive events from n = 3 independent color-coded experiments is shown. Two-tailed t test. ns, not significant. ( D ) NIH3T3 cells stably expressing nAC-tagGFP were transfected with siCtrl or siRNA targeting INF2 or SUN2 and stimulated with 0.4 U/mL thrombin to induce rapid nuclear actin assembly. In total 453 (siCtrl), 417 (siINF2), 390 (siSUN2 #1) or 500 (siSUN2 #2) cells were scored from n = 4 independent color-coded experiments. One-way ANOVA with Dunnett’s multiple comparison test, P < 0.0001. ( E ) Immunoblots showing siRNA-mediated emerin, SUN2, or INF2 knockdown efficiency. ( F ) Immunoblots showing siRNA-mediated nesprin1, nesprin2, or nesprin3 knockdown efficiency. ( G ) NIH3T3 cells stably expressing nAC-tagGFP were transfected with mScarlet-tagged dominant-negative KASH (dnKASH-mSc) or mScarlet (mSc) empty vector and stimulated with 1 μM A23187 to induce rapid nuclear actin assembly. In total, 415 (mSc) or 441 (dnKASH-mSc) cells were scored from n = 4 independent experiments. Mann–Whitney test. ns not significant. ( H ) NIH3T3 cells stably expressing nAC-tagGFP were transfected with siCtrl or siRNA targeting nesprin1, nesprin2, and nesprin3 and stimulated with 1 μM A23187 to induce rapid nuclear actin assembly. In total, 267 (siCtrl) or 213 (siSyne1-3) cells were scored from n = 3 independent experiments. Two-tailed t test. ns not significant. Data information: In ( C , D , G , H ), data are represented as mean ± SD. .

Article Snippet: Primary antibodies used were: mAb to α-tubulin (1:1000, CST, no. 2125), mAb to emerin (1:500, CST, no. 30853), pAb to INF2 (1:500, Proteintech, no. 20466-1-AP), mAb to SUN2 (1:1000, Abcam, no. ab124916), mAb to Flag (DYKDDDDK) tag (1:1000, CST, no. 14793), mAb to Syne3 (1:1000, OriGene, no. AM33013PU-N), pAb to Syne2 (1:1000, Invitrogen, no. PA5-78438), mAb to Syne1 (1:1000, Abcam, ab192234).

Techniques: Stable Transfection, Expressing, Membrane, Dominant Negative Mutation, Transfection, Control, Two Tailed Test, Comparison, Western Blot, Knockdown, Plasmid Preparation, MANN-WHITNEY

Journal: EMBO Reports

Article Title: SUN2 mediates calcium-triggered nuclear actin polymerization to cluster active RNA polymerase II

doi: 10.1038/s44319-024-00274-8

Figure Lengend Snippet: ( A ) Cellular distribution of detected PLA signals. Each bar represents mean +/− SEM from at least three independent experiments. ( B ) Quantification of cytosolic PLA signal reveals no significant increase in number upon A2387 treatment. Data are shown as violin plot with median and quartiles. Individual data points represent one field of view acquired from three independent biological replicates. two-tailed t test. ns, not significant. ( C ) Representative MIPs of NIH3T3 cells subjected to PLA and quantified in Fig. . Discrete PLA signals (Cy5) indicate the interaction of endogenous target proteins. Scale bar = 20 µm. ( D ) Proximity ligation assay (PLA) of NIH3T3 cells using the indicated primary antibodies. PLA probes alone serve as technical control to detect nonspecific binding of PLA probes. Use of SUN2 primary antibody together with antibodies directed against the known interactors Lamin A/C or emerin serve as positive control. Primary antibody against cytoplasmic tubulin serves as negative biological control. Images show representative maximum-intensity projections (MIP) of 20 confocal z-slices (0.21 μm z-distance). Discrete PLA signals (Cy5) indicate the interaction of endogenous target proteins within the nucleus (DAPI). Scale bar = 20 μm. ( E ) Quantification of PLA signal (dots/nucleus) from technical controls using indicated primary antibodies alone. Data are shown as violin plot with median and quartiles. Individual data points represent one field of view acquired from three independent biological replicates. ( F ) Orthogonal optical cross section reconstructed from a Z-scan through a NIH3T3 cell nucleus stained for DAPI and PLA dots (Cy5) between SUN2 and emerin or SUN2 and Lamin A/C. Scale bar = 5 μm. ( G ) Immunoblot showing siRNA-mediated SUN2 knockdown efficiency of cells subjected to PLA (Fig. ).

Article Snippet: Primary antibodies used were: mAb to α-tubulin (1:1000, CST, no. 2125), mAb to emerin (1:500, CST, no. 30853), pAb to INF2 (1:500, Proteintech, no. 20466-1-AP), mAb to SUN2 (1:1000, Abcam, no. ab124916), mAb to Flag (DYKDDDDK) tag (1:1000, CST, no. 14793), mAb to Syne3 (1:1000, OriGene, no. AM33013PU-N), pAb to Syne2 (1:1000, Invitrogen, no. PA5-78438), mAb to Syne1 (1:1000, Abcam, ab192234).

Techniques: Two Tailed Test, Proximity Ligation Assay, Control, Binding Assay, Positive Control, Staining, Western Blot, Knockdown

Journal: EMBO Reports

Article Title: SUN2 mediates calcium-triggered nuclear actin polymerization to cluster active RNA polymerase II

doi: 10.1038/s44319-024-00274-8

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Primary antibodies used were: mAb to α-tubulin (1:1000, CST, no. 2125), mAb to emerin (1:500, CST, no. 30853), pAb to INF2 (1:500, Proteintech, no. 20466-1-AP), mAb to SUN2 (1:1000, Abcam, no. ab124916), mAb to Flag (DYKDDDDK) tag (1:1000, CST, no. 14793), mAb to Syne3 (1:1000, OriGene, no. AM33013PU-N), pAb to Syne2 (1:1000, Invitrogen, no. PA5-78438), mAb to Syne1 (1:1000, Abcam, ab192234).

Techniques: Recombinant, Sequencing, Negative Control, Transfection, Protease Inhibitor, Membrane, In Situ, Software, Microscopy, Laser-Scanning Microscopy

Journal: Pathology and Oncology Research

Article Title: Negative correlation between the nuclear size and nuclear Lamina component Lamin A in intraductal papillary mucinous neoplasms of the pancreas

doi: 10.3389/pore.2022.1610684

Figure Lengend Snippet: Primary antibodies used.

Article Snippet: Emerin , CL0201 , Mouse monoclonal , 2 μg/ml , NBP2-52876, Novus Biologicals, Centennial, CO, US.

Techniques: Concentration Assay