progerin cdna  (Addgene inc)

Journal: Cells

Article Title: Progerin Expression Induces Inflammation, Oxidative Stress and Senescence in Human Coronary Endothelial Cells

doi: 10.3390/cells9051201

Figure Lengend Snippet: Expression and localization of exogenous lamin A and progerin. Early-confluent human coronary artery endothelial cells (HCAECs) were transduced or not with Flag-tagged recombinant adenovirus containing WT-prelamin A or progerin for 72 h, or with an empty vector (AdNul). ( A ) Western blot analysis of lamin A and C and progerin. β-actin was used as a loading control. Representative picture of n = 4 experiments. ( B ) Representative pictures of transduced HCAECs stained with Flag (green) and lamin-A (red) antibodies. Cells were observed by confocal microscopy at 100× magnification. Examples of misshapen nuclei are indicated with a white arrow. ( C ) Quantification of misshapen nuclei as percentage of total nuclei. Data are expressed as the mean ± standard error of mean (SEM) and statistical difference is determined using analysis of variance (ANOVA) followed by a Dunnett post hoc test. *** p < 0.001 vs. WT.

Article Snippet: Δ50 prelamin A (progerin) cDNA was obtained from GeneArt (Thermo Fisher scientific, Invitrogen Corporation, San Diego, CA, USA) from full-length rat prelamin A cDNA. cDNA of WT-prelamin A or progerin was integrated in a pAd5 plasmid vector, under the control of the cytomegalovirus (CMV) promoter.

Techniques: Expressing, Recombinant, Plasmid Preparation, Western Blot, Control, Staining, Confocal Microscopy

Journal: Cells

Article Title: Progerin Expression Induces Inflammation, Oxidative Stress and Senescence in Human Coronary Endothelial Cells

doi: 10.3390/cells9051201

Figure Lengend Snippet: Progerin induces endothelial cells inflammation and dysfunction. Early-confluent HCAECs were transduced or not with Flag-tagged recombinant adenovirus containing WT-prelamin A or progerin for 72 h or with an empty vector. ( A ) Twenty-four hour secretion of the proinflammatory cytokines IL6, IL1β, of the chemokines CXCL8 and CCL2 and of the adhesion molecules ICAM1 and VCAM1. ( B ) Adhesion assay of peripheral blood mononuclear cells (PBMCs) from healthy donors on transduced and control endothelial cells was quantified as the number of adherent PBMC/mg of protein (left panel). Representative pictures are shown in the right panel with a magnification of the white squared area ( C ) Relative mRNA expression of the endothelial nitric oxide synthase gene ( NOS3 ). Data are expressed as the mean ± SEM and statistical difference is determined using ANOVA followed by a Dunnett post hoc test. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. WT.

Article Snippet: Δ50 prelamin A (progerin) cDNA was obtained from GeneArt (Thermo Fisher scientific, Invitrogen Corporation, San Diego, CA, USA) from full-length rat prelamin A cDNA. cDNA of WT-prelamin A or progerin was integrated in a pAd5 plasmid vector, under the control of the cytomegalovirus (CMV) promoter.

Techniques: Recombinant, Plasmid Preparation, Cell Adhesion Assay, Control, Expressing

Journal: Cells

Article Title: Progerin Expression Induces Inflammation, Oxidative Stress and Senescence in Human Coronary Endothelial Cells

doi: 10.3390/cells9051201

Figure Lengend Snippet: Endothelial progerin expression induces oxidative stress, DNA damage and cellular senescence. Early-confluent HCAECs were transduced or not with Flag-tagged recombinant adenovirus containing WT-prelamin A or progerin or with an empty vector. ( A ) Reactive oxygen species (ROS) production was assessed by the oxidation of 5-6-chloromethyl-2,7-dichlorodihydro-fluorescein diacetate (CM-H2DCFDA). ( B ) Relative mRNA expression of DDIT3 . ( C ) DNA double-strand breaks (DSBs) were studied by staining HCAECs with Ser139-phosphorylated histone variant H2A (γ-H2AX, in green) and di-amidino-2-phenylindole hydrochloride (DAPI) (in blue) (upper panel) and evaluated as the percentage of γ-H2AX–positive cells (40–200 cells per experiment) (lower panel). ( D ) Protein expression of the cell cycle arrest proteins p53 and p21. Representative picture of n = 3 experiments. ( E ) Senescence-associated (SA)-β-galactosidase activity was assessed by the percentage of 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal)-stained HCAECs (in blue) at pH6. Representative micrographs are shown (upper panel). Data are expressed as the mean ± SEM and statistical difference determined using ANOVA followed by a Dunnett post hoc test. ** p < 0.01, *** p < 0.001 vs. WT.

Article Snippet: Δ50 prelamin A (progerin) cDNA was obtained from GeneArt (Thermo Fisher scientific, Invitrogen Corporation, San Diego, CA, USA) from full-length rat prelamin A cDNA. cDNA of WT-prelamin A or progerin was integrated in a pAd5 plasmid vector, under the control of the cytomegalovirus (CMV) promoter.

Techniques: Expressing, Recombinant, Plasmid Preparation, Staining, Variant Assay, Activity Assay

Journal: Cells

Article Title: Progerin Expression Induces Inflammation, Oxidative Stress and Senescence in Human Coronary Endothelial Cells

doi: 10.3390/cells9051201

Figure Lengend Snippet: Inhibition of progerin prenylation partially prevents endothelial cells senescence, inflammation and secretion of adhesion molecules. Early-confluent HCAECs were transduced or not with Flag-tagged recombinant adenovirus containing WT-prelamin A or progerin for 72 h and immediately treated with zoledronate and pravastatin (ZOPRA). ( A ) DNA DSBs were studied by staining HCAECs with γ-H2AX (in green) and DAPI (in blue). Representative micrographs of HCAECs expressing progerin are shown (left panel). DNA DSBs were quantified as the percentage of γ-H2AX–positive cells (40–200 cells per experiment) (right panel). ( B ) Senescence-associated (SA)-β-galactosidase activity was assessed as the percentage of X-gal–stained cells (blue) at pH6 (right panel). Representative micrographs are shown (left panel). ( C ) 24 h-secretion of the proinflammatory cytokine IL6, of the chemokines CXCL8 and CCL2 and of the adhesion molecules ICAM1 and VCAM1. Data are expressed as the mean ± SEM and statistical difference was determined using two-way ANOVA followed by a Tukey post hoc test to assess differences against WT-prelamin A overexpression or a Sidak post hoc test to determine ZOPRA effect for each condition. *** p < 0.001 vs. WT. ## p < 0.01, ### p < 0.001 vs. vehicle-treated cells.

Article Snippet: Δ50 prelamin A (progerin) cDNA was obtained from GeneArt (Thermo Fisher scientific, Invitrogen Corporation, San Diego, CA, USA) from full-length rat prelamin A cDNA. cDNA of WT-prelamin A or progerin was integrated in a pAd5 plasmid vector, under the control of the cytomegalovirus (CMV) promoter.

Techniques: Inhibition, Recombinant, Staining, Expressing, Activity Assay, Over Expression

Journal: Scientific Reports

Article Title: Expression of the Hutchinson-Gilford Progeria Mutation Leads to Aberrant Dentin Formation

doi: 10.1038/s41598-018-33764-6

Figure Lengend Snippet: Induction of DNA damage and cellular senescence by progerin expression in odontoblasts. ( a ) Progerin-elicited structural changes in the nuclear envelope of MDPC-23 cells with Δ50 lamin A were compared to the WT lamin A and the negative control. ( b ) Percentage of cells with abnormal nucleus was compared by scoring blebs or invaginations along the membrane. ( c , d ) Phosphorylated H2AX-positive nuclei in MDPC-23 with Δ50 lamin A were detected by immunofluorescence staining using antibodies for γH2AX ( c ) and their counted percentage ( d ) compared to the WT lamin A and the negative control. ( e ) Confirmation of increased DNA damage in MDPC-23 with Δ50 lamin A by Western blot analysis using antibodies for human lamin A (LA), γH2AX and phosphorylated p53 (p-p53). The samples shown were derived from the same experiment, and all gels/blots were processed under the same experimental conditions. β-actin was used as a loading control. Cropped images are displayed here; the original full-size blots are presented in Supplementary Fig. . ( f ) Growth rates of MDPC-23 cells with Δ50 lamin A, WT lamin A and the negative control were compared until day 8 in serum-free media. Significance was assigned for p -values as indicated. Bars , 5 μm ( a and c ).

Article Snippet: Plasmids driving the expression of GFP-tagged human lamin A and the truncated exon 1–12 encoding the cDNA for progerin (Δ50 lamin A) were gifts from Tom Misteli (Addgene plasmids #17662 and #17663, respectively).

Techniques: Expressing, Negative Control, Membrane, Immunofluorescence, Staining, Western Blot, Derivative Assay, Control

Journal: Scientific Reports

Article Title: Expression of the Hutchinson-Gilford Progeria Mutation Leads to Aberrant Dentin Formation

doi: 10.1038/s41598-018-33764-6

Figure Lengend Snippet: Defects in signaling pathways and downregulation of matrix protein expression in progerin-expressing odontoblasts. ( a ) Promoter activities for BMP and TGFβ signaling were analyzed and compared in MDPC-23 transfected with Δ50 lamin A, WT lamin A and the negative control by luciferase reporters of BRE and SBE, respectively. ( b ) Promoter activities for Wnt/β-catenin signaling were analyzed and compared in MDPC-23 transfected with Δ50 lamin A, WT lamin A and the negative control by luciferase reporters of FOPflash/TOPflash. ( c ) Western blot of cytoplasmic and nuclear protein was performed using antibodies for nonphosphor-β-catenin (Active β-Cat), β-catenin (β-Cat), histone H3 and α-tubulin. Progerin expression by transfection of G608G lamin A and Δ50 lamin A impairs nuclear translocation of β-catenin. The samples shown were derived from the same experiment, and all gels/blots were processed under the same experimental conditions. α-tubulin and histone H3 were used as a loading control for cytoplasmic and nuclear protein, respectively. Cropped images are displayed here; the original full-size blots are presented in Supplementary Fig. . ( d ) The transcript levels of dentin matrix-related genes in MDPC-23 transfected with Δ50 lamin A, WT lamin A and the negative control were analyzed and compared by real-time qPCR. Significance was assigned for p -values as indicated.

Article Snippet: Plasmids driving the expression of GFP-tagged human lamin A and the truncated exon 1–12 encoding the cDNA for progerin (Δ50 lamin A) were gifts from Tom Misteli (Addgene plasmids #17662 and #17663, respectively).

Techniques: Protein-Protein interactions, Expressing, Transfection, Negative Control, Luciferase, Western Blot, Translocation Assay, Derivative Assay, Control

Journal: Scientific Reports

Article Title: Expression of the Hutchinson-Gilford Progeria Mutation Leads to Aberrant Dentin Formation

doi: 10.1038/s41598-018-33764-6

Figure Lengend Snippet: Paracrine factors induced by progerin stimulate odontogenic differentiation of dental pulp cells. ( a ) The transcript levels of genes for soluble factors were analyzed by real-time qPCR of MDPC-23 transfected with Δ50 lamin A, WT lamin A and the negative control. ( b , c ) Mineralization ability of dental pulp cells treated with conditioned media from MDPC-23 cells of Δ50 lamin A (Δ50-CM), WT lamin A (WT-CM) and the negative control (Control-CM) was exhibited by alizarin red staining ( b ) and evaluated ( c ). ( d ) The transcript levels of odontogenesis-related genes were analyzed by real-time qPCR with dental pulp cells treated with conditioned media of Δ50 lamin A (Δ50-CM), WT lamin A (WT-CM) and the negative control (Control-CM) for 2 days. Significance was assigned for p -values as indicated. Bars , 40 μm ( b ).

Article Snippet: Plasmids driving the expression of GFP-tagged human lamin A and the truncated exon 1–12 encoding the cDNA for progerin (Δ50 lamin A) were gifts from Tom Misteli (Addgene plasmids #17662 and #17663, respectively).

Techniques: Transfection, Negative Control, Control, Staining

Journal: Scientific Reports

Article Title: Expression of the Hutchinson-Gilford Progeria Mutation Leads to Aberrant Dentin Formation

doi: 10.1038/s41598-018-33764-6

Figure Lengend Snippet: Ctgf induced by progerin stimulates odontogenic differentiation of dental pulp cells. ( a ) The expression of Ctgf in odontoblasts was analyzed with whole cell extract from MDPC-23 transfected with Δ50 lamin A, WT lamin A and the negative control by Western blot analysis using an antibody for Ctgf. The samples shown were derived from the same experiment, and all gels/blots were processed under the same experimental conditions. β-actin was used as a loading control. Cropped images are displayed here; the original full-size blots are provided in Supplementary Fig. . ( b ) Reduced mineralization of mouse dental pulp cells by neutralization of Ctgf using polyclonal anti-Ctgf IgG. Alizarin red staining was performed with mouse dental pulp cells treated with conditioned media from the culture of MDPC-23 cells expressing Δ50 lamin A (Δ50-CM) and WT lamin A (WT-CM) supplemented with polyclonal anti-Ctgf IgG (2 μg/ml) and control IgG for 2 days. ( c , d ) Mineralization ability of dental pulp cells treated with different amounts of recombinant Ctgf peptide was exhibited by alizarin red staining ( c ) and evaluated ( d ). Significance was assigned for p -values as indicated. Bars , 40 μm ( c ).

Article Snippet: Plasmids driving the expression of GFP-tagged human lamin A and the truncated exon 1–12 encoding the cDNA for progerin (Δ50 lamin A) were gifts from Tom Misteli (Addgene plasmids #17662 and #17663, respectively).

Techniques: Expressing, Transfection, Negative Control, Western Blot, Derivative Assay, Control, Neutralization, Staining, Recombinant

Journal: Scientific Reports

Article Title: Expression of the Hutchinson-Gilford Progeria Mutation Leads to Aberrant Dentin Formation

doi: 10.1038/s41598-018-33764-6

Figure Lengend Snippet: Schematic working model proposing the major hypothesis. During initial dentinogenesis in WT, odontoblasts form a primary dentin until the tooth becomes functional. When contacts between antagonistic cusps are established, then the formation of secondary dentin starts immediately, and continues throughout life. However, in HGPS mice, progerin-induced cellular senescence of odontoblasts occurred by progerin results in impaired physiological secondary dentin formation, which is an intermediate outcome. Physiologically, this thinner dentin may be recognized as an injury, such as dentin trauma. Subsequently, odontoblast-like cells differentiated from dental pulp cells might rapidly produce tertiary dentin until the pulp is completely obliterated in the HGPS mice. Paracrine factors released from odontoblasts undergoing senescence accelerate tertiary dentin formation by stimulating odontogenic differentiation of pulp cells.

Article Snippet: Plasmids driving the expression of GFP-tagged human lamin A and the truncated exon 1–12 encoding the cDNA for progerin (Δ50 lamin A) were gifts from Tom Misteli (Addgene plasmids #17662 and #17663, respectively).

Techniques: Functional Assay