Journal: Journal of Clinical Investigation

Article Title: Endothelial progerin expression causes cardiovascular pathology through an impaired mechanoresponse

doi: 10.1172/jci121297

Figure Lengend Snippet: Figure 1. Characterization of Prog-Tg mice. (A) Immunoblots of indicated lysates using anti– human lamin A/C (detecting transgenes, upper panel), anti–lamin A/C (recognizing human and mouse lamin A/C, lower panel), and anti-tubu- lin and anti–VE-cadherin as loading controls. Numbers show ratio of total lamin A levels in transgenic over Wt animals. Endothelial cells (ECs) from Wt (Wt-EC), Prog-Tg (Prog-Tg-EC) and LA-Tg (LA-Tg-EC) animals; EC-depleted cell mix- ture from Prog-Tg animals (Prog-Tg-non-EC); and HGPS patient fibroblasts (HGPS) were analyzed. (B) Histogram of mean progerin fluorescence intensities in immunofluorescence images of Prog-Tg ECs (see Figure 5C) (n = 5 Prog-Tg mice, 462 cells in total). (C and D) Immunofluorescence images of coronary artery (C) and cardiac tissue (D) from Prog-Tg animals stained with antibodies against progerin, VE-cadherin (note green aut- ofluorescence of elastic lamina), and PECAM1, and Hoechst (representative of n = 3 Prog-Tg animals). Progerin expression is confined to the intimal layer (C, arrowheads) and PECAM1-pos- itive cardiac microvasculature (D, arrowheads). Dashed lines, cardiomyocyte boundaries; arrows, intima (C) and progerin-negative cardiomyocytes (D). Scale bars: 10 μm. (E) Body weight over time for male and female Prog-Tg and LA-Tg versus Wt littermates. Two-way repeated-measures ANOVA revealed a significant impact for the Prog-Tg genotype (females F = 72.6, P < 0.001, males F = 65.3, P < 0.001, n = 6 littermate pairs), but not for the LA-Tg genotype (females F = 0.517, P = 0.493; males F = 0.221, P = 0.651, n = 5 littermate pairs). Comparison of Prog-Tg versus Wt revealed at least P < 0.01 (Holm-Sidak meth- od) for females and males at more than 5 and 8 weeks, respectively. (F) Kaplan-Meier survival plot showing significantly reduced life span of Prog-Tg mice (n = 12) versus Wt littermate con- trols (n = 20) and LA-Tg mice (n = 8). P < 0.0001, log-rank (Mantel-Cox) test; pairwise comparison with Bonferroni’s correction of threshold showed significant difference in survival of Prog-Tg compared with control, Prog-Tg with LA-Tg, but not LA-Tg with control mice. Data presented as mean ± SEM.

Article Snippet: The following primary antibodies were used: mouse monoclonal antibody (mAb) against lamin A/C (Santa Cruz Biotechnology, clone E-1, sc-376248), mAb against human lamin A+C (Chemicon, clone JoL2, mab3211, Abcam), goat anti-MRTFA (Santa Cruz Biotechnology, sc-21558), mAb against progerin (Alexis Biochemicals, clone 13A4, provided by Egon Ogris, Medical University Vienna, Vienna, Austria), mAb against α-tubulin (MilliporeSigma, clone B-5-1-2, T5168), mAb against emerin (Leica Biosystems, clone 4G5, NCL-emerin), rabbit anti-emerin (for immunoblotting, Atlas Antibodies, HPA000609), goat anti–VE-cadherin antibodies (Santa Cruz Biotechnology, sc-6458), rabbit anti-PECAM1 (LSBio, LS-B4737), mAb against eNOS (BD Biosciences, 610297), rabbit affinity-purified antibodies against actin (MilliporeSigma, A2066), guinea pig anti-SUN1 and -SUN2 antibodies (a gift from Manfred Alsheimer, University of Würzburg, Würzburg, Germany).

Techniques: Western Blot, Transgenic Assay, Fluorescence, Immunofluorescence, Staining, Expressing, Comparison, Control

Journal: Journal of Clinical Investigation

Article Title: Endothelial progerin expression causes cardiovascular pathology through an impaired mechanoresponse

doi: 10.1172/jci121297

Figure Lengend Snippet: Figure 7. Impaired MRTFA signaling in Prog-Tg ECs affects eNOS and profibrotic signaling. (A) Immunoflu- orescence of Wt and Prog-Tg ECs using MRTFA antibody and DAPI (images are representative of n = 3 inde- pendent experiments). Scale bar: 10 μm. (B) Representative confocal average intensity projections of Z stacks from mouse aorta of Wt and Prog-Tg animals stained with DAPI and MRTFA and progerin antibodies. EC nuclei (arrowheads) lie on internal elastic membrane that displays blue autofluorescence. Arrowheads, MRTFA-positive ECs. Note that MRTFA accumulates at the nuclear periphery of progerin-positive (white arrowheads) but not progerin-negative (yellow arrowheads) ECs (n = 3 Wt and Prog-Tg littermate pairs). Scale bar: 10 μm. (C) Nos3 mRNA in Wt and Prog-Tg ECs after 24-hour treatment with 15 μM CCG-203971 MRTFA inhibitor or DMSO vehicle control. Values are normalized to Hprt and Nos3/Hprt values in Prog-Tg cells are shown relative to values in Wt cells after MRTFA-inhibitor and control treatment. Nos3/Hprt levels in Wt cells were arbitrary and set to 1 in both control and CCG-203971 conditions (n = 5 independent experiments). Nos3/Hprt levels in Prog-Tg cells relative to Wt are not significantly different (NS) after drug treatment, in contrast to control conditions (P < 0.01). (D) Chromatin immunoprecipitation using anti-MRTFA or goat IgG control. Precipitated DNA was amplified by qPCR with primers spanning Nos3 promoter or gene body (n = 3 independent experiments). **P < 0.01 by unpaired Stu- dent’s t test (C and D). Data presented as mean ± SEM. (E) Acta2 levels normalized to Hprt in fibroblasts after 3 days of coculture with Wt and Prog-Tg ECs either left untreated (left) or treated with 25 μM MRTFA inhibitor CCG-203971 (right). Values were subtracted from those obtained in untreated or CCG-203971–treated fibroblast single cultures (n = 6 Wt, n = 8 Prog-Tg, and n = 3 inhibitor-treated Prog-Tg samples). *P < 0.05 by Mann-Whitney U test. Data presented as median (middle line) with boxes encompassing 25th to 75th percentile, and whiskers, minimum to maximum values.

Article Snippet: The following primary antibodies were used: mouse monoclonal antibody (mAb) against lamin A/C (Santa Cruz Biotechnology, clone E-1, sc-376248), mAb against human lamin A+C (Chemicon, clone JoL2, mab3211, Abcam), goat anti-MRTFA (Santa Cruz Biotechnology, sc-21558), mAb against progerin (Alexis Biochemicals, clone 13A4, provided by Egon Ogris, Medical University Vienna, Vienna, Austria), mAb against α-tubulin (MilliporeSigma, clone B-5-1-2, T5168), mAb against emerin (Leica Biosystems, clone 4G5, NCL-emerin), rabbit anti-emerin (for immunoblotting, Atlas Antibodies, HPA000609), goat anti–VE-cadherin antibodies (Santa Cruz Biotechnology, sc-6458), rabbit anti-PECAM1 (LSBio, LS-B4737), mAb against eNOS (BD Biosciences, 610297), rabbit affinity-purified antibodies against actin (MilliporeSigma, A2066), guinea pig anti-SUN1 and -SUN2 antibodies (a gift from Manfred Alsheimer, University of Würzburg, Würzburg, Germany).

Techniques: Staining, Membrane, Control, Chromatin Immunoprecipitation, Amplification, MANN-WHITNEY

Journal: Journal of Clinical Investigation

Article Title: Endothelial progerin expression causes cardiovascular pathology through an impaired mechanoresponse

doi: 10.1172/jci121297

Figure Lengend Snippet: Figure 8. Working model. Progerin accumulation at the nuclear lamina leads to perturbations in F-actin level and actin organization and defects in components involved in nucleocytoskeletal coupling (SUN1/2 and emerin). The defective rigid nucleocytoskeletal links show impaired mechanoresponse and cause MRTFA accumulation at the nuclear periphery. MRTFA or associated complexes exert presumably a positive feedback loop on Actb expression (looped arrow). These changes set off the profibrotic signaling cascade (indicated by pale orange color of the nucleus), such as downregulation of eNOS and reduced secretion of NO. Absence of endothelium-derived atheroprotective NO and presumably other unidentified factors leads to increased collagen production in other cell types as shown for fibroblasts through a switch to myofibroblasts, leading to collagen deposition and cardiac fibrosis.

Article Snippet: The following primary antibodies were used: mouse monoclonal antibody (mAb) against lamin A/C (Santa Cruz Biotechnology, clone E-1, sc-376248), mAb against human lamin A+C (Chemicon, clone JoL2, mab3211, Abcam), goat anti-MRTFA (Santa Cruz Biotechnology, sc-21558), mAb against progerin (Alexis Biochemicals, clone 13A4, provided by Egon Ogris, Medical University Vienna, Vienna, Austria), mAb against α-tubulin (MilliporeSigma, clone B-5-1-2, T5168), mAb against emerin (Leica Biosystems, clone 4G5, NCL-emerin), rabbit anti-emerin (for immunoblotting, Atlas Antibodies, HPA000609), goat anti–VE-cadherin antibodies (Santa Cruz Biotechnology, sc-6458), rabbit anti-PECAM1 (LSBio, LS-B4737), mAb against eNOS (BD Biosciences, 610297), rabbit affinity-purified antibodies against actin (MilliporeSigma, A2066), guinea pig anti-SUN1 and -SUN2 antibodies (a gift from Manfred Alsheimer, University of Würzburg, Würzburg, Germany).

Techniques: Expressing, Derivative Assay

Journal: PLoS ONE

Article Title: Upregulation of the aging related LMNA splice variant progerin in dilated cardiomyopathy

doi: 10.1371/journal.pone.0196739

Figure Lengend Snippet: A Schematic overview of PCR based strategy to analyze progerin mRNA expression in human heart and the blood samples. Shown are the consensus donor splice sequence at the end of exon 11, the sequence of the normal LMNA cryptic splice site, and two common known mutated cryptic splice sites in HGPS patients. B First row: PCR gel showing total lamin A (first arrow) and small amounts of progerin expression (arrow showing Δ150bp band). Second row: Specific progerin expression utilizing primers spanning exon 11 to 12. Third row: RPL32 expression was used for relative quantification of progerin expression. G: Genomic DNA (G) did not reveal any significant LMNA bands in the gel verifying specific mRNA expression.

Article Snippet: For Immunofluorescence staining, deparaffinized sections were incubated with primary mouse monoclonal antibodies against progerin (ab66587, Abcam) or (sc-81611, Santa Cruz) overnight at 4°C followed by a one-hour incubation with secondary antibody at room temperature (Alexa Fluor® 488, Molecular Probes Inc, Eugene, OR) and wheat germ agglutinin (WGA) Texas RedTM-X Conjugate (W21405, Invitrogen).

Techniques: Expressing, Sequencing, Quantitative Proteomics

Journal: PLoS ONE

Article Title: Upregulation of the aging related LMNA splice variant progerin in dilated cardiomyopathy

doi: 10.1371/journal.pone.0196739

Figure Lengend Snippet: The sequence of purified progerin PCR cDNA (red letters) was aligned to the genomic sequence of LMNA exon 11 (green) to exon 12 (yellow) with the 3´UTR sequence (blue) showing the expected gap of 150bp between exon 11 to exon 12 confirming alternative splicing of progerin.

Article Snippet: For Immunofluorescence staining, deparaffinized sections were incubated with primary mouse monoclonal antibodies against progerin (ab66587, Abcam) or (sc-81611, Santa Cruz) overnight at 4°C followed by a one-hour incubation with secondary antibody at room temperature (Alexa Fluor® 488, Molecular Probes Inc, Eugene, OR) and wheat germ agglutinin (WGA) Texas RedTM-X Conjugate (W21405, Invitrogen).

Techniques: Sequencing, Purification, Alternative Splicing

Journal: PLoS ONE

Article Title: Upregulation of the aging related LMNA splice variant progerin in dilated cardiomyopathy

doi: 10.1371/journal.pone.0196739

Figure Lengend Snippet: A Relative amount of progerin mRNA levels related to the reference gene RPL32 in heart biopsies of patients with DCM (n = 15) and non-failing control hearts (n = 10). Shown are all individual data points, lines show mean ± SD. ** P ≤ 0.01 DCM vs. Control. B Relative amount of progerin mRNA levels related to the reference gene RPL32 in whole blood cells derived from patients with DCM (n = 56) and healthy controls (n = 10). Shown are all individual data points, lines show mean ± SD. P-value was derived from unpaired two-sided T-Test between the groups. C Scatter plot showing the negative correlation between the relative amount of progerin mRNA related to RPL32 with ejection fraction in human heart biopsies (n = 25). ** P ≤ 0.01. D Scatter plot showing the positive correlation between the relative amount of progerin mRNA related to RPL32 with left ventricular enddiastolic diameter (LVEDD) and E Scatter plot showing the positive correlation between progerin mRNA related to RPL32 with the age of the hearts in human heart biopsies (n = 25). N.s. not significant. ** P ≤ 0.01.

Article Snippet: For Immunofluorescence staining, deparaffinized sections were incubated with primary mouse monoclonal antibodies against progerin (ab66587, Abcam) or (sc-81611, Santa Cruz) overnight at 4°C followed by a one-hour incubation with secondary antibody at room temperature (Alexa Fluor® 488, Molecular Probes Inc, Eugene, OR) and wheat germ agglutinin (WGA) Texas RedTM-X Conjugate (W21405, Invitrogen).

Techniques: Control, Derivative Assay

Journal: PLoS ONE

Article Title: Upregulation of the aging related LMNA splice variant progerin in dilated cardiomyopathy

doi: 10.1371/journal.pone.0196739

Figure Lengend Snippet: Progerin and inflammation in the heart.

Article Snippet: For Immunofluorescence staining, deparaffinized sections were incubated with primary mouse monoclonal antibodies against progerin (ab66587, Abcam) or (sc-81611, Santa Cruz) overnight at 4°C followed by a one-hour incubation with secondary antibody at room temperature (Alexa Fluor® 488, Molecular Probes Inc, Eugene, OR) and wheat germ agglutinin (WGA) Texas RedTM-X Conjugate (W21405, Invitrogen).

Techniques:

Journal: PLoS ONE

Article Title: Upregulation of the aging related LMNA splice variant progerin in dilated cardiomyopathy

doi: 10.1371/journal.pone.0196739

Figure Lengend Snippet: A Immunohistochemistry showing increased numbers of progerin expressing cells (arrows, red nuclei) in DCM hearts compared to non-failing controls. Scale bar represents 100μM (rows 1–2) and 25μM (rows 3–4). B Immunofluorescence stainings revealed increased expression of progerin (green, arrows) in nuclei (blue) of DCM hearts (merged images last row, arrows) compared to non-failing controls. Red: WGA membrane staining, Green: Progerin staining, Blue: DAPI+ nuclei. Scale bar represents 20μM. C TUNEL staining of apoptotic cell death revealed increased numbers of TUNEL+ cells in DCM hearts compared to non-failing controls. Scale bar represents 200μM (rows 1–3) and 20μM (last row). D Quantification of progerin+ nuclei to total nuclei per high power field (HPF) in DCM hearts and Controls (n = 3). ** P ≤ 0.01 DCM vs. Control. E Quantification of apoptotic index (TUNEL+ nuclei to total nuclei) in DCM hearts compared to controls (n = 3). ** P ≤ 0.01 DCM vs. Control.

Article Snippet: For Immunofluorescence staining, deparaffinized sections were incubated with primary mouse monoclonal antibodies against progerin (ab66587, Abcam) or (sc-81611, Santa Cruz) overnight at 4°C followed by a one-hour incubation with secondary antibody at room temperature (Alexa Fluor® 488, Molecular Probes Inc, Eugene, OR) and wheat germ agglutinin (WGA) Texas RedTM-X Conjugate (W21405, Invitrogen).

Techniques: Immunohistochemistry, Expressing, Immunofluorescence, Membrane, Staining, TUNEL Assay, Control

Journal: PLoS ONE

Article Title: Upregulation of the aging related LMNA splice variant progerin in dilated cardiomyopathy

doi: 10.1371/journal.pone.0196739

Figure Lengend Snippet: Immunofluorescence stainings revealed expression of the apoptotic cell death marker cleaved caspase-3 (red, arrow) and progerin (green, arrow) in the nucleus (blue) of a cardiomyocyte delineated by red cell membrane staining with WGA (merged image last row, arrow) compared to non-failing controls. Scale bar represents 20μM.

Article Snippet: For Immunofluorescence staining, deparaffinized sections were incubated with primary mouse monoclonal antibodies against progerin (ab66587, Abcam) or (sc-81611, Santa Cruz) overnight at 4°C followed by a one-hour incubation with secondary antibody at room temperature (Alexa Fluor® 488, Molecular Probes Inc, Eugene, OR) and wheat germ agglutinin (WGA) Texas RedTM-X Conjugate (W21405, Invitrogen).

Techniques: Immunofluorescence, Expressing, Marker, Membrane, Staining

Journal: Cell Death & Disease

Article Title: Angiomotin-p130 inhibits β-catenin stability by competing with Axin for binding to tankyrase in breast cancer

doi: 10.1038/s41419-019-1427-2

Figure Lengend Snippet: a Protein levels of total β-catenin, cytoplasmic β-catenin, and nuclear β-catenin as determined by western blotting. GAPDH was used as the loading control for the total and cytoplasmic protein. Lamin A was used as the loading control for nuclear protein. b β-Catenin-driven transcription activity was determined by TOP/FOP luciferase reporter assays. Normalization was based on internal Renilla luciferase actvity. The final reporter activity was measured as TOP/FOP ratio and was expressed as the mean ± SD of three independent experiments. c Protein levels of WNT downstream targets were determined by western blotting. d β-Catenin expression was determined by immunohistochemistry staining in xenograft tissues. Scale bar = 200 µm. e Co-localization of Amot-p130 (red) and β-catenin (green) in cell–cell contacts was indicated by immunofluorescence confocal microscopy. DAPI was used for nuclear staining (blue). f The interaction between Amot-p130 and β-catenin was evaluated in MCF7 cells by co-immunoprecipitation assay. IgG was used as the negative control. *** P < 0.001. DAPI, 4′,6-diamidino-2-phenylin

Article Snippet: HRP-labeled GAPDH (HRP-60004) and antibody against lamin A (10298-1-AP) were from Proteintech Group.

Techniques: Western Blot, Activity Assay, Luciferase, Expressing, Immunohistochemistry, Staining, Immunofluorescence, Confocal Microscopy, Co-Immunoprecipitation Assay, Negative Control

Journal: Cell Death & Disease

Article Title: Angiomotin-p130 inhibits β-catenin stability by competing with Axin for binding to tankyrase in breast cancer

doi: 10.1038/s41419-019-1427-2

Figure Lengend Snippet: a β-Catenin levels in cells treated with CHX (200 µg/ml) for the indicated time, with or without MG132 (20 µM) for 2 h, were determined using western blotting (left). GAPDH was used as the loading control. The curve showed the relative trend of β-catenin changes (right). b β-Catenin levels in cells treated with XAV939 10 µg/ml for 24 h were determined using western blotting (left). The quantitation of β-catenin was expressed as the mean ± SD of three independent experiments (right). c β-catenin levels in cells treated with SKL2001 30 µM for 24 h were determined using western blotting (left). The quantitation of β-catenin was expressed as the mean ± SD of three independent experiments (right). d Protein levels of the total, cytoplasmic, and nuclear β-catenin in cells treated with XAV939 or SKL2001, alone or in combination with MG132, were determeined using western blotting. GAPDH was used as the loading control for the total and cytoplasmic protein. Lamin A was used as the loading control for nuclear protein. e MCF7 cell proliferation under XAV939 or SKL2001 treatment was determined by MTT assay. f Relative quantitation of Amot-p130 and Axin pulled down by TNKS in co-immunoprecipitation assay. IgG pull-down was used as the negative control. * P < 0.05, ** P < 0.01, *** P < 0.001; ns, no significance. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TNKS, tankyrase

Article Snippet: HRP-labeled GAPDH (HRP-60004) and antibody against lamin A (10298-1-AP) were from Proteintech Group.

Techniques: Western Blot, Quantitation Assay, MTT Assay, Co-Immunoprecipitation Assay, Negative Control