Journal: Life Science Alliance

Article Title: A novel role for CSA in the regulation of nuclear envelope integrity: uncovering a non-canonical function

doi: 10.26508/lsa.202402745

Figure Lengend Snippet: (A) Representative Western blot of the indicated NE proteins in WT(HA-CSA) and CS-A cells. (B) Representative immunofluorescence images of lamin A/C, lamin B1, emerin, and SUN1 staining; scale bar: 25 μm. (C) Representative immunofluorescence staining showing total and insoluble LEMD2 protein in WT(HA-CSA) and CS-A cells; scale bar: 25 μm. (D) Quantification of the insoluble LEMD2 showing a significant reduction in CS-A compared with WT(HA-CSA) using a two-tailed unpaired t test (** P < 0.01), n = 3 with >100 cells per experiment. (E) Representative immunoblot showing LEMD2 expression level in the soluble, insoluble, and whole cell extracts in WT(HA-CSA) and CS-A cells. (E, F) Quantification of the relative LEMD2 expression level from the western blots as shown in (E); normalized to GAPDH, histone H3, and α-tubulin, respectively. P -values were calculated using a two-tailed paired t tests (ns P > 0.05, * P < 0.05). All experiments in this figure were n = 3 independent experiments. Source data are available for this figure.

Article Snippet: Primary antibodies for incubation were mouse anti-lamin A/C (#sc-376248, 1:1,000; Santa Cruz) and rabbit anti-LEMD2 (#HPA017340, 1:250; Sigma-Aldrich).

Techniques: Western Blot, Immunofluorescence, Staining, Two Tailed Test, Expressing

Journal: Life Science Alliance

Article Title: A novel role for CSA in the regulation of nuclear envelope integrity: uncovering a non-canonical function

doi: 10.26508/lsa.202402745

Figure Lengend Snippet: Representative immunofluorescence images of LEMD2 in pre-extracted WT(HA-CSA) and CS-A cells following siRNA-mediated knockdown of LEMD2 (siLEMD2); scale bar: 50 μm.

Article Snippet: Primary antibodies for incubation were mouse anti-lamin A/C (#sc-376248, 1:1,000; Santa Cruz) and rabbit anti-LEMD2 (#HPA017340, 1:250; Sigma-Aldrich).

Techniques: Immunofluorescence, Knockdown

Journal: Life Science Alliance

Article Title: A novel role for CSA in the regulation of nuclear envelope integrity: uncovering a non-canonical function

doi: 10.26508/lsa.202402745

Figure Lengend Snippet: (A) Representative immunofluorescence staining showing insoluble LEMD2 protein in WT AG10803 and CSA KO AG10803 cells; scale bar: 25 μm. (C) Quantification of the insoluble LEMD2 showing a significant reduction in CSA KO AG10803 compared with WT AG10803 cells using a two-tailed unpaired t test (** P < 0.01), n = 3 with >100 cells per experiment. (B) Representative immunofluorescence staining showing the appearance of F-actin stress fibers in CS-A cells compared with WT(HA-CSA), scale bar: 25 μm.

Article Snippet: Primary antibodies for incubation were mouse anti-lamin A/C (#sc-376248, 1:1,000; Santa Cruz) and rabbit anti-LEMD2 (#HPA017340, 1:250; Sigma-Aldrich).

Techniques: Immunofluorescence, Staining, Two Tailed Test

Journal: Life Science Alliance

Article Title: A novel role for CSA in the regulation of nuclear envelope integrity: uncovering a non-canonical function

doi: 10.26508/lsa.202402745

Figure Lengend Snippet: (A) Representative confocal images of DAPI and PLA signal in the indicated cells using either anti-lamin A/C or anti-LEMD2 antibodies alone (negative controls) or in combination; scale bar: 50 μm. (B) Quantification of the number of PLA foci per nuclei. P -value was calculated using a one-way ANOVA test followed by Tukey’s post hoc test (**** P < 0.0001), n = 3 with >100 cells per experiment. (C) Representative immunofluorescence staining of DAPI and lamin B1 in WT(HA-CSA) and CS-A cells transfected with GFP and LEMD2-GFP-containing constructs. (D, E) Quantification of the nuclear form factor and (E) percentage of nuclear blebs in GFP expressing cells, n = 3 with >100 cells per experiment. P -value was calculated using a one-way ANOVA test followed by Tukey’s post hoc test (* P < 0.05, *** P < 0.001, **** P < 0.0001). All experiments in this figure were n = 3 independent experiments.

Article Snippet: Primary antibodies for incubation were mouse anti-lamin A/C (#sc-376248, 1:1,000; Santa Cruz) and rabbit anti-LEMD2 (#HPA017340, 1:250; Sigma-Aldrich).

Techniques: Immunofluorescence, Staining, Transfection, Construct, Expressing

Journal: Life Science Alliance

Article Title: A novel role for CSA in the regulation of nuclear envelope integrity: uncovering a non-canonical function

doi: 10.26508/lsa.202402745

Figure Lengend Snippet: (A) Immunoprecipitation of LEMD2-GFP in WT(HA-CSA) cells overexpressing GFP+Flag-CSA (control) and Flag-CSA+LEMD2-GFP. (B) Representative time-lapse confocal images showing pre-bleach, bleach, and post-bleach images of the WT(HA-CSA) and CS-A nuclei with LEMD2-GFP overexpression. The red box indicates the photobleached region at the nuclear periphery. (C) Graph showing the FRAP kinetics of LEMD2-GFP expressed in WT(HA-CSA) (n = 25 cells) and CS-A (n = 25 cells) cells. Error bars represent SD. Scale bar: 5 μm. (D) Summary table showing the mean percentage of the immobile fraction and the mean half-time of recovery of LEMD2-GFP FRAP experiment. Data were compared using a two-tailed unpaired t test. All experiments in this figure were n = 3 independent experiments. Source data are available for this figure.

Article Snippet: Primary antibodies for incubation were mouse anti-lamin A/C (#sc-376248, 1:1,000; Santa Cruz) and rabbit anti-LEMD2 (#HPA017340, 1:250; Sigma-Aldrich).

Techniques: Immunoprecipitation, Control, Over Expression, Two Tailed Test

Journal: Life Science Alliance

Article Title: A novel role for CSA in the regulation of nuclear envelope integrity: uncovering a non-canonical function

doi: 10.26508/lsa.202402745

Figure Lengend Snippet: Details of primary and secondary antibodies used for immunofluorescence and Western blot experiments.

Article Snippet: Primary antibodies for incubation were mouse anti-lamin A/C (#sc-376248, 1:1,000; Santa Cruz) and rabbit anti-LEMD2 (#HPA017340, 1:250; Sigma-Aldrich).

Techniques: Immunofluorescence, Western Blot

Journal: Life Science Alliance

Article Title: A novel role for CSA in the regulation of nuclear envelope integrity: uncovering a non-canonical function

doi: 10.26508/lsa.202402745

Figure Lengend Snippet: (A) Representative Western blot of the indicated NE proteins in WT(HA-CSA) and CS-A cells. (B) Representative immunofluorescence images of lamin A/C, lamin B1, emerin, and SUN1 staining; scale bar: 25 μm. (C) Representative immunofluorescence staining showing total and insoluble LEMD2 protein in WT(HA-CSA) and CS-A cells; scale bar: 25 μm. (D) Quantification of the insoluble LEMD2 showing a significant reduction in CS-A compared with WT(HA-CSA) using a two-tailed unpaired t test (** P < 0.01), n = 3 with >100 cells per experiment. (E) Representative immunoblot showing LEMD2 expression level in the soluble, insoluble, and whole cell extracts in WT(HA-CSA) and CS-A cells. (E, F) Quantification of the relative LEMD2 expression level from the western blots as shown in (E); normalized to GAPDH, histone H3, and α-tubulin, respectively. P -values were calculated using a two-tailed paired t tests (ns P > 0.05, * P < 0.05). All experiments in this figure were n = 3 independent experiments. Source data are available for this figure.

Article Snippet: Anti-LEMD2 , 1:200 , 1:200 , Sigma-Aldrich , HPA017340.

Techniques: Western Blot, Immunofluorescence, Staining, Two Tailed Test, Expressing

Journal: Life Science Alliance

Article Title: A novel role for CSA in the regulation of nuclear envelope integrity: uncovering a non-canonical function

doi: 10.26508/lsa.202402745

Figure Lengend Snippet: Representative immunofluorescence images of LEMD2 in pre-extracted WT(HA-CSA) and CS-A cells following siRNA-mediated knockdown of LEMD2 (siLEMD2); scale bar: 50 μm.

Article Snippet: Anti-LEMD2 , 1:200 , 1:200 , Sigma-Aldrich , HPA017340.

Techniques: Immunofluorescence, Knockdown

Journal: Life Science Alliance

Article Title: A novel role for CSA in the regulation of nuclear envelope integrity: uncovering a non-canonical function

doi: 10.26508/lsa.202402745

Figure Lengend Snippet: (A) Representative immunofluorescence staining showing insoluble LEMD2 protein in WT AG10803 and CSA KO AG10803 cells; scale bar: 25 μm. (C) Quantification of the insoluble LEMD2 showing a significant reduction in CSA KO AG10803 compared with WT AG10803 cells using a two-tailed unpaired t test (** P < 0.01), n = 3 with >100 cells per experiment. (B) Representative immunofluorescence staining showing the appearance of F-actin stress fibers in CS-A cells compared with WT(HA-CSA), scale bar: 25 μm.

Article Snippet: Anti-LEMD2 , 1:200 , 1:200 , Sigma-Aldrich , HPA017340.

Techniques: Immunofluorescence, Staining, Two Tailed Test

Journal: Life Science Alliance

Article Title: A novel role for CSA in the regulation of nuclear envelope integrity: uncovering a non-canonical function

doi: 10.26508/lsa.202402745

Figure Lengend Snippet: (A) Representative confocal images of DAPI and PLA signal in the indicated cells using either anti-lamin A/C or anti-LEMD2 antibodies alone (negative controls) or in combination; scale bar: 50 μm. (B) Quantification of the number of PLA foci per nuclei. P -value was calculated using a one-way ANOVA test followed by Tukey’s post hoc test (**** P < 0.0001), n = 3 with >100 cells per experiment. (C) Representative immunofluorescence staining of DAPI and lamin B1 in WT(HA-CSA) and CS-A cells transfected with GFP and LEMD2-GFP-containing constructs. (D, E) Quantification of the nuclear form factor and (E) percentage of nuclear blebs in GFP expressing cells, n = 3 with >100 cells per experiment. P -value was calculated using a one-way ANOVA test followed by Tukey’s post hoc test (* P < 0.05, *** P < 0.001, **** P < 0.0001). All experiments in this figure were n = 3 independent experiments.

Article Snippet: Anti-LEMD2 , 1:200 , 1:200 , Sigma-Aldrich , HPA017340.

Techniques: Immunofluorescence, Staining, Transfection, Construct, Expressing

Journal: Life Science Alliance

Article Title: A novel role for CSA in the regulation of nuclear envelope integrity: uncovering a non-canonical function

doi: 10.26508/lsa.202402745

Figure Lengend Snippet: (A) Immunoprecipitation of LEMD2-GFP in WT(HA-CSA) cells overexpressing GFP+Flag-CSA (control) and Flag-CSA+LEMD2-GFP. (B) Representative time-lapse confocal images showing pre-bleach, bleach, and post-bleach images of the WT(HA-CSA) and CS-A nuclei with LEMD2-GFP overexpression. The red box indicates the photobleached region at the nuclear periphery. (C) Graph showing the FRAP kinetics of LEMD2-GFP expressed in WT(HA-CSA) (n = 25 cells) and CS-A (n = 25 cells) cells. Error bars represent SD. Scale bar: 5 μm. (D) Summary table showing the mean percentage of the immobile fraction and the mean half-time of recovery of LEMD2-GFP FRAP experiment. Data were compared using a two-tailed unpaired t test. All experiments in this figure were n = 3 independent experiments. Source data are available for this figure.

Article Snippet: Anti-LEMD2 , 1:200 , 1:200 , Sigma-Aldrich , HPA017340.

Techniques: Immunoprecipitation, Control, Over Expression, Two Tailed Test

Journal: Life Science Alliance

Article Title: A novel role for CSA in the regulation of nuclear envelope integrity: uncovering a non-canonical function

doi: 10.26508/lsa.202402745

Figure Lengend Snippet: Details of primary and secondary antibodies used for immunofluorescence and Western blot experiments.

Article Snippet: Anti-LEMD2 , 1:200 , 1:200 , Sigma-Aldrich , HPA017340.

Techniques: Immunofluorescence, Western Blot

Journal: Frontiers in Oncology

Article Title: The expression and role of the Lem-D proteins Ankle2, Emerin, Lemd2, and TMPO in triple-negative breast cancer cell growth

doi: 10.3389/fonc.2024.1222698

Figure Lengend Snippet: Expression of several Lem-D proteins is significantly elevated in TNBC tumor patient samples. (A–D) Box plots of Lem-D gene expression comparing normal and breast tumor patient samples utilizing the Gene Expression database of Normal and Tumor tissues 2 (GENT2) database: (A) Ankle2 expression, (B) TMPO expression, (C) Emerin expression, and (D) Lemd2 expression. (E–H) Graphical representation of expression of the Lem proteins in Stages I, II, and III TNBC patient samples utilizing The Cancer Genetics Atlas (TCGA) database: (E) Ankle2 (F) TMPO (G) Emerin, and (H) Lemd2 expressions. (I–L) Kaplan–Meier values for Lem-D gene expression in breast cancer patients showed that high mRNA expression of Lem-D genes was associated with a decrease in the probability of patient overall survival. Lem-D mRNA transcript expression was categorized as high or low expressed based on the median mRNA expression within the database. The effect of (I) Ankle2, (J) TMPO, (K) Emerin, and (L) Lemd2 mRNA expression on patient overall survival. For (A–H) : error bars denote standard deviation of the mean. Statistical significance was calculated using a Kruskal–Wallis test: **** p < 0.0001, ** p < 0.0021. For (I–L) : statistical significance was calculated using a log-rank p -test. ns, not significant.

Article Snippet: Antibodies used were as follows: anti-Emerin (5430, Cell Signaling Technology, Danvers, MA, USA 1:500 for IF, 1:1000 for IB), anti-Lemd2 (PA553589, Thermo Fisher Scientific, Waltham MA, USA 1:300 for IF, 1:1000 for IB), anti-Ankle2 (GTX120698, Genetex, Irvine, CA, United States 1:200 for IF, 1:1000 for IB), and anti-TMPO (L3414-.2ML, Sigma-Aldrich, Saint Louis, MO 1:500 for IF, 1:1000 for IB), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (D16H11, Cell Signaling Technology, 1:4000 for IB), and anti–Gamma-Tubulin (T6557, Sigma-Aldrich, 1:3000 for IB).

Techniques: Expressing, Standard Deviation

Journal: Frontiers in Oncology

Article Title: The expression and role of the Lem-D proteins Ankle2, Emerin, Lemd2, and TMPO in triple-negative breast cancer cell growth

doi: 10.3389/fonc.2024.1222698

Figure Lengend Snippet: Expression of the Lem-domain proteins in TNBC cells. (A) Representative Western blot of BT549, Hs578T, MDA-MB-231, and MDA-MB-468 whole cell lysates showing expression of Lemd2, Ankle2, Emerin, and TMPO isoforms in TNBC cell lines, in comparison to the control MCF10A non-cancerous breast tissue cells. β−Actin was utilized as a loading control to allow for standardization via densitometry in ImageJ Software (B–G) Graphs represent densitometry analysis of Lem protein expression determined via Western blot analysis in TNBC normalized to non-cancerous MCF10A cells: (B) Ankle2 (C) Lemd2 (D) Emerin, and (E–G) TMPOα, β, and γ. Graphed values represent results from three individual repeats and error bars denote standard deviation of the mean. Statistical significance was calculated using an unpaired t-test: *** p < 0.0002, ** p < 0.0021, * p < 0.0332.

Article Snippet: Antibodies used were as follows: anti-Emerin (5430, Cell Signaling Technology, Danvers, MA, USA 1:500 for IF, 1:1000 for IB), anti-Lemd2 (PA553589, Thermo Fisher Scientific, Waltham MA, USA 1:300 for IF, 1:1000 for IB), anti-Ankle2 (GTX120698, Genetex, Irvine, CA, United States 1:200 for IF, 1:1000 for IB), and anti-TMPO (L3414-.2ML, Sigma-Aldrich, Saint Louis, MO 1:500 for IF, 1:1000 for IB), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (D16H11, Cell Signaling Technology, 1:4000 for IB), and anti–Gamma-Tubulin (T6557, Sigma-Aldrich, 1:3000 for IB).

Techniques: Expressing, Western Blot, Comparison, Software, Standard Deviation

Journal: Frontiers in Oncology

Article Title: The expression and role of the Lem-D proteins Ankle2, Emerin, Lemd2, and TMPO in triple-negative breast cancer cell growth

doi: 10.3389/fonc.2024.1222698

Figure Lengend Snippet: The Lem-D proteins are consistently localized in TNBC cells. (A) Representative immunofluorescent microscopy images of MCF10A, BT549, Hs578T, MDA-MB-231, and MDA-MB-468 cells. Cells were stained with Ankle2, TMPO, Emerin, and Lemd2 antibodies. Cells were counterstained with Hoechst 33342 (blue). (B–E) Quantification of the portion of cells with individual Lem-D proteins localized to the NE. (B) Ankle2, (C) TMPO, (D) Emerin, and (E) Lemd2. Quantifications are based on 200 cells/condition in at least three experimentally independent experiments. Error bars denote standard deviation of the mean. Scale bars = 10 µM. Statistical significance was calculated using an unpaired t-test: * p < 0.0332.

Article Snippet: Antibodies used were as follows: anti-Emerin (5430, Cell Signaling Technology, Danvers, MA, USA 1:500 for IF, 1:1000 for IB), anti-Lemd2 (PA553589, Thermo Fisher Scientific, Waltham MA, USA 1:300 for IF, 1:1000 for IB), anti-Ankle2 (GTX120698, Genetex, Irvine, CA, United States 1:200 for IF, 1:1000 for IB), and anti-TMPO (L3414-.2ML, Sigma-Aldrich, Saint Louis, MO 1:500 for IF, 1:1000 for IB), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (D16H11, Cell Signaling Technology, 1:4000 for IB), and anti–Gamma-Tubulin (T6557, Sigma-Aldrich, 1:3000 for IB).

Techniques: Microscopy, Staining, Standard Deviation

Journal: Frontiers in Oncology

Article Title: The expression and role of the Lem-D proteins Ankle2, Emerin, Lemd2, and TMPO in triple-negative breast cancer cell growth

doi: 10.3389/fonc.2024.1222698

Figure Lengend Snippet: siRNA-mediated depletion of the Lem-D proteins induces aberrant NE morphology in TNBC cells. Representative immunofluorescent microscopy images of MCF10A and TNBC cells transfected with control and Lem-domain (Lem-D) protein siRNAs. Cells were stained with Hoechst 33342 to visualize the nucleus. Representative cells categorized to have normal and abnormal nuclear morphology: (A) MCF10A (B) BT549 (C) Hs578T (D) MDA-MB-231, and (E) MDA-MB-468. Quantification of cells with aberrant nuclear morphology in Control and Lem-D protein siRNA transfected cells. Normalized nuclear form factor values for: (F) Ankle2, (G) TMPO, (H) Emerin, and (I) Lemd2 siRNA. Form Factor score of 1 = perfect round nucleus. Quantifications are based on 200 cells/condition in at least three independent experiments. Error bars denote standard deviation of the mean. Statistical significance was calculated using an unpaired t-test: **** p < 0.0001, *** p < 0.0002, ** p < 0.0021, * p < 0.0332. ns, not significant.

Article Snippet: Antibodies used were as follows: anti-Emerin (5430, Cell Signaling Technology, Danvers, MA, USA 1:500 for IF, 1:1000 for IB), anti-Lemd2 (PA553589, Thermo Fisher Scientific, Waltham MA, USA 1:300 for IF, 1:1000 for IB), anti-Ankle2 (GTX120698, Genetex, Irvine, CA, United States 1:200 for IF, 1:1000 for IB), and anti-TMPO (L3414-.2ML, Sigma-Aldrich, Saint Louis, MO 1:500 for IF, 1:1000 for IB), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (D16H11, Cell Signaling Technology, 1:4000 for IB), and anti–Gamma-Tubulin (T6557, Sigma-Aldrich, 1:3000 for IB).

Techniques: Microscopy, Transfection, Staining, Standard Deviation

Journal: Frontiers in Oncology

Article Title: The expression and role of the Lem-D proteins Ankle2, Emerin, Lemd2, and TMPO in triple-negative breast cancer cell growth

doi: 10.3389/fonc.2024.1222698

Figure Lengend Snippet: siRNA-mediated depletion of the Lem-D proteins induces TNBC cell death. Graphs represent percent of live, apoptotic (early and late apoptotic) and necrotic cells 5 days post-transfection with Control, Ankle2, TMPO, Emerin, and Lemd2 siRNAs (A) MCF10A (B) BT549 (C) MDA-MB-231 cells. Graphed values represent results from three individual repeats and error bars denote standard deviation of the mean. Statistical significance was calculated using an unpaired t -test: **** p < 0.0001, *** p < 0.0002. ns, not significant.

Article Snippet: Antibodies used were as follows: anti-Emerin (5430, Cell Signaling Technology, Danvers, MA, USA 1:500 for IF, 1:1000 for IB), anti-Lemd2 (PA553589, Thermo Fisher Scientific, Waltham MA, USA 1:300 for IF, 1:1000 for IB), anti-Ankle2 (GTX120698, Genetex, Irvine, CA, United States 1:200 for IF, 1:1000 for IB), and anti-TMPO (L3414-.2ML, Sigma-Aldrich, Saint Louis, MO 1:500 for IF, 1:1000 for IB), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (D16H11, Cell Signaling Technology, 1:4000 for IB), and anti–Gamma-Tubulin (T6557, Sigma-Aldrich, 1:3000 for IB).

Techniques: Transfection, Standard Deviation

Journal: The Journal of Clinical Investigation

Article Title: Loss of function of the nuclear envelope protein LEMD2 causes DNA damage–dependent cardiomyopathy

doi: 10.1172/JCI158897

Figure Lengend Snippet: ( A ) WT and Lemd2 c.T38>G (KI) alleles showing the sgRNA and the protospacer adjacent motif (PAM) sequences as well as the introduced pathogenic (red) and silent (green) mutations. ( B ) Survival curve of WT ( n = 17), KI/+ ( n = 22), and KI/KI ( n = 15) mice. **** P < 0.0001 for WT versus KI/KI comparison, log-rank (Mantel-Cox) test. ( C ) Western blot showing the levels of 2 cardiac LEMD2 protein isoforms in 2-month-old WT ( n = 4) and KI/KI ( n = 4) mice. Bottom: average and SEM of the relative LEMD2/GAPDH densitometry ratio in WT and KI/KI mice. ( D ) Macroscopic images of hearts from 3-month-old WT and KI/KI mice. Scale bar: 0.5 cm. RA, right atrium; LA, left atrium; RV, right ventricle. ( E ) H&E staining of hearts of 3-month-old WT and KI/KI mice. Scale bar: 500 μm. ( F ) Masson’s trichrome staining of 4-chamber view hearts from 3-month-old WT and KI/KI mice. Scale bar: 500 μm.

Article Snippet: The mouse LEMD2 ORF was purchased from Addgene (plasmid 120245) and cloned into the previously generated AAV9 vector under the control of the cTnT promoter ( ).

Techniques: Comparison, Western Blot, Staining

Journal: The Journal of Clinical Investigation

Article Title: Loss of function of the nuclear envelope protein LEMD2 causes DNA damage–dependent cardiomyopathy

doi: 10.1172/JCI158897

Figure Lengend Snippet: ( A ) Representative photograph of Lemd2 fl/fl (left) and cKO (right) mice at P7. Scale bar: 1 cm. ( B ) Survival curve of Lemd2 fl/fl ( n = 19) and cKO ( n = 23) mice. **** P < 0.0001, log-rank (Mantel-Cox) test. ( C ) Echocardiographic analysis of systolic EF of hearts from Lemd2 fl/fl ( n = 7) and cKO ( n = 3) mice (from 2 to 10 postnatal days).** P < 0.01, 2-tailed, unpaired t test. ( D ) Echocardiographic analysis of systolic FS of hearts from Lemd2 fl/fl ( n = 7) and cKO ( n = 3) mice (from 2 to 10 postnatal days). ** P < 0.01, 2-tailed, unpaired t test. ( E ) H&E staining of 4-chamber view P7 hearts from Lemd2 fl/fl and cKO mice. Scale bar: 500 μm. Note the atrial thrombi in cKO hearts. ( F ) Heatmap showing the DE genes in Lemd2 fl/fl and cKO mice. n = 3 mice per genotype. Expression level in Z -score. FDR-adjusted P value of 0.05 was used as cutoff, Benjamini–Hochberg method. ( G ) Enriched GO terms up- and downregulated in cKO compared with Lemd2 fl/fl mice. n = 3 mice per genotype. P < 0.01. ( H ) Upstream regulator analysis of DE genes in cKO mice compared with Lemd2 fl/fl mice. n = 4 mice per genotype. Activation in Z -score.

Article Snippet: The mouse LEMD2 ORF was purchased from Addgene (plasmid 120245) and cloned into the previously generated AAV9 vector under the control of the cTnT promoter ( ).

Techniques: Staining, Expressing, Activation Assay

Journal: The Journal of Clinical Investigation

Article Title: Loss of function of the nuclear envelope protein LEMD2 causes DNA damage–dependent cardiomyopathy

doi: 10.1172/JCI158897

Figure Lengend Snippet: ( A ) Schematic representation of the confiner device. ( B ) Percentage of nuclei positive for γ-H2AX in Lemd2 fl/fl and cKO CMs isolated from P1 mice at baseline and after 20 μm compression for 1 hour. Three experimental replicates. n = 2–5 mice per genotype, more than 80 nuclei per replicate. *** P < 0.001; ** P < 0.01; * P < 0.05, 1-way ANOVA and Holm-Šidák test for correction of multiple comparisons. ( C ) Representative images of γ-H2AX and cTNT staining in CMs isolated from P1 Lemd2 fl/fl and cKO mice at baseline and after 20 μm compression for 1 hour. Scale bar: 20 μm. ( D ) Percentage of TUNEL-positive nuclei in Lemd2 fl/fl and cKO CMs isolated from P1 mice at baseline and after 20 μm compression for 1 hour. 2–3 replicates. n = 2–5 mice per genotype, more than 80 nuclei per replicate. ** P < 0.01; * P < 0.05, 1-way ANOVA and Holm-Šidák test for correction of multiple comparisons. ( E ) Representative images of TUNEL and cTnI staining in Lemd2 fl/fl and cKO CMs isolated from P1 mice and compressed at 20 μm for 1 hour. Scale bar: 20 μm. ( F ) Nuclei area in CMs isolated from P1 Lemd2 fl/fl and cKO mice at baseline and after 20 μm compression for 1 hour. n = 2 mice per genotype, 15–30 nuclei per genotype. * P < 0.05, 1-way ANOVA and Holm-Šidák test for correction of multiple comparisons. ( G ) Nuclear solidity (area/convex area) in CMs isolated from P1 Lemd2 fl/fl and cKO mice at baseline and after 20 μm compression for 1 hour. n = 2 mice per genotype, 15–30 nuclei per genotype. *** P < 0.001; * P < 0.05, 1-way ANOVA and Holm-Šidák test for correction of multiple comparisons. ( H ) Representative images of lamin B1 and cTnI staining in Lemd2 fl/fl and cKO CMs isolated from P1 mice and compressed at 20 μm for 1 hour. Scale bar: 5 μm.

Article Snippet: The mouse LEMD2 ORF was purchased from Addgene (plasmid 120245) and cloned into the previously generated AAV9 vector under the control of the cTnT promoter ( ).

Techniques: Isolation, Staining, TUNEL Assay

Journal: The Journal of Clinical Investigation

Article Title: Loss of function of the nuclear envelope protein LEMD2 causes DNA damage–dependent cardiomyopathy

doi: 10.1172/JCI158897

Figure Lengend Snippet: ( A ) Schematic of the AAV9- Lemd2 system for in vivo delivery. ( B ) Overview of the in vivo injection strategy. ( C – G ) Echocardiographic analysis of structural and functional parameters in hearts from 2-month-old WT ( n = 7), KI/KI ( n = 10), and KI/KI AAV9- Lemd2 mice ( n = 4). The AAV9- Lemd2 treatment experiment was unblinded for mouse genotypes, and data are compared with untreated WT and KI/KI groups shown in , that were not assessed contemporaneously. ( C ) Systolic LVAW thickness, ( D ) systolic LVID, ( E ) EF, ( F ) FS, and ( G ) LV volume. ( H ) H&E staining of 4-chamber view of 3-month-old hearts from WT, KI/KI, and KI/KI AAV9- Lemd2 mice. Scale bar: 500 μm. ( I ) Masson trichrome staining of 3-month-old hearts from WT, KI/KI, and KI/KI AAV9- Lemd2 mice. Scale bar: 50 μm. ( J ) Quantification of the percentage of cardiac fibrosis in hearts from WT, KI/KI, and KI/KI AAV9- Lemd2 mice. 4–5 cardiac sections per mouse. n = 1 mouse per genotype. ** P < 0.01; * P < 0.05, 1-way ANOVA and Holm-Šidák test for correction of multiple comparisons. ( K ) Lemd2 mRNA expression in hearts from 2-month-old WT ( n = 4), KI/KI ( n = 4), and KI/KI AAV9- Lemd2 ( n = 3) mice. *** P < 0.001, 1-way ANOVA and Holm-Šidák test for correction of multiple comparisons. ( L ) Western blot showing the levels of 2 cardiac LEMD2 protein isoforms in 2-month-old WT ( n = 3), KI/KI ( n = 3), and KI/KI AAV9- Lemd2 ( n = 3) mice. Bottom: average and SEM of the relative LEMD2/GAPDH densitometry ratio in WT, KI/KI, and KI/KI AAV9- Lemd2 mice.

Article Snippet: The mouse LEMD2 ORF was purchased from Addgene (plasmid 120245) and cloned into the previously generated AAV9 vector under the control of the cTnT promoter ( ).

Techniques: In Vivo, Injection, Functional Assay, Staining, Expressing, Western Blot