plasmid plpc hrap1 fl  (Addgene inc)

Journal: Metabolism: clinical and experimental

Article Title: Endothelial senescence-associated secretory phenotype (SASP) is regulated by Makorin-1 ubiquitin E3 ligase

doi: 10.1016/j.metabol.2019.153962

Figure Lengend Snippet: (A, B) HUVECs were transfected with TERF2IP, MKRN1, or control siRNA in combinations as shown for 48 h and transduced with Ad-GFP or Ad-p90RSK-WT for 12 h. The cells were then assayed for apoptosis by quantifying FITC annexin V-positive cells (A). Telomere lengths were measured as described in the Methods (B). Data represent mean ± SD (n = 3–6). Cell viability was assessed by the MTT colorimetric assay as described in the Methods (C). Data present mean ± SD (n = 3). (D) HUVECs were transfected with TERF2IP, MKRN1, or control siRNA in combinations as indicated together with NF-κB reporter gene for 48 h and transduced with Ad-GFP or Ad-p90RSK-WT for 12 h. NF-κB activity was assayed by the dual-luciferase reporter assay. Data represent mean ± SD (n = 6). **p <0.01. All data were analyzed by one-way ANOVA followed by the Bonferroni post hoc test.

Article Snippet: Plasmids containing rat wild type (WT) p90RSK1 (WT- p90rsk ) (Genebank NM031107) was generated as we previously described[ 19 ]. pLPC-human TERF2IP full length (# 12542 ) was from Addgene. pCMV-Flag-TERF2IP-WT was obtained by subcloning TERF2IP from pLPC-human TERF2IP full length into the pCMV-Tag2B vector (Agilent Technologies, Santa Clara, CA) at sites recognized by the restriction enzymes Eco RI and Xho I.

Techniques: Transfection, Control, Transduction, Colorimetric Assay, Activity Assay, Luciferase, Reporter Assay

Journal: Metabolism: clinical and experimental

Article Title: Endothelial senescence-associated secretory phenotype (SASP) is regulated by Makorin-1 ubiquitin E3 ligase

doi: 10.1016/j.metabol.2019.153962

Figure Lengend Snippet: (A, B) HUVECs were transfected with MKRN1 or control siRNA for 48 h and transduced with Ad-GFP or Ad-TERF2IP S205A for 12 h. The cells were then assayed for apoptosis by quantifying FITC annexin V-positive cells (A). Telomere lengths were measured as described in the Methods (B). Data represent mean ± SD (n = 3–6). (C) HUVECs were transfected with MKRN1 or control siRNA and also with NF-κB reporter gene for 48 h and transduced with Ad-GFP or Ad-TERF2IP S205A for 12 h. NF-κB activity was assayed by the dual-luciferase reporter assay. Data represent mean ± SD (n = 6). **p <0.01. All data were analyzed by one-way ANOVA followed by the Bonferroni post hoc test.

Article Snippet: Plasmids containing rat wild type (WT) p90RSK1 (WT- p90rsk ) (Genebank NM031107) was generated as we previously described[ 19 ]. pLPC-human TERF2IP full length (# 12542 ) was from Addgene. pCMV-Flag-TERF2IP-WT was obtained by subcloning TERF2IP from pLPC-human TERF2IP full length into the pCMV-Tag2B vector (Agilent Technologies, Santa Clara, CA) at sites recognized by the restriction enzymes Eco RI and Xho I.

Techniques: Transfection, Control, Transduction, Activity Assay, Luciferase, Reporter Assay

Journal: Metabolism: clinical and experimental

Article Title: Endothelial senescence-associated secretory phenotype (SASP) is regulated by Makorin-1 ubiquitin E3 ligase

doi: 10.1016/j.metabol.2019.153962

Figure Lengend Snippet: (A, B) Gene and miRNA expression profiles regulated by TERF2IP and d-flow were determined by hierarchical clustering with p = 6.6e-5 (A) or p = 3.3e-5 (B). The gene abbreviations are defined in Supplementary Table 1. (C) The principal component analysis, based on the conditions of TERF2IP depletion by siRNA and d-flow, was performed at a stringent p = 3.3e-5. (D) HAECs were transfected by incubation with control siRNA or TERF2IP siRNA for 48 h and subjected to d-flow for 16 h. Expression of RICTOR and MKRN1 mRNAs was determined by quantitative real-time PCR. Data represent mean ± SD (n = 4; **p<0.01, *p<0.05. All data were analyzed by one-way ANOVA followed by the Bonferroni post hoc test.

Article Snippet: Plasmids containing rat wild type (WT) p90RSK1 (WT- p90rsk ) (Genebank NM031107) was generated as we previously described[ 19 ]. pLPC-human TERF2IP full length (# 12542 ) was from Addgene. pCMV-Flag-TERF2IP-WT was obtained by subcloning TERF2IP from pLPC-human TERF2IP full length into the pCMV-Tag2B vector (Agilent Technologies, Santa Clara, CA) at sites recognized by the restriction enzymes Eco RI and Xho I.

Techniques: Expressing, Transfection, Incubation, Control, Real-time Polymerase Chain Reaction

Journal: Metabolism: clinical and experimental

Article Title: Endothelial senescence-associated secretory phenotype (SASP) is regulated by Makorin-1 ubiquitin E3 ligase

doi: 10.1016/j.metabol.2019.153962

Figure Lengend Snippet: Since we found no difference of d-flow-induced SASP induction between HAECs and HUVECs, we used HUVECs to study the mechanisms of d-flow-induced SASP in Fig.2–5. HUVECs were transfected with control, TERF2IP, or MKRN1 siRNA in combinations as shown for 48 h and transduced with Ad-GFP or Ad-p90RSK-WT for 12 h. Western blotting was performed with specific antibodies as indicated (A, C). The graphs represent densitometry data from immunoblots. Mean ± SD (n = 3) (B, D). **p<0.01.

Article Snippet: Plasmids containing rat wild type (WT) p90RSK1 (WT- p90rsk ) (Genebank NM031107) was generated as we previously described[ 19 ]. pLPC-human TERF2IP full length (# 12542 ) was from Addgene. pCMV-Flag-TERF2IP-WT was obtained by subcloning TERF2IP from pLPC-human TERF2IP full length into the pCMV-Tag2B vector (Agilent Technologies, Santa Clara, CA) at sites recognized by the restriction enzymes Eco RI and Xho I.

Techniques: Transfection, Control, Transduction, Western Blot

Journal: Metabolism: clinical and experimental

Article Title: Endothelial senescence-associated secretory phenotype (SASP) is regulated by Makorin-1 ubiquitin E3 ligase

doi: 10.1016/j.metabol.2019.153962

Figure Lengend Snippet: HUVECs were transfected with control siRNA or MKRN1 siRNA for 48 h and transduced with Ad-GFP or Ad-TERF2IP S205A for 12 h. Expression of indicated proteins was detected by Western blotting performed using specific antibodies (A, C). Graphs represent densitometry data from immunoblots. Data represent mean ± SD (n = 3) (B, D). **p <0.01.

Article Snippet: Plasmids containing rat wild type (WT) p90RSK1 (WT- p90rsk ) (Genebank NM031107) was generated as we previously described[ 19 ]. pLPC-human TERF2IP full length (# 12542 ) was from Addgene. pCMV-Flag-TERF2IP-WT was obtained by subcloning TERF2IP from pLPC-human TERF2IP full length into the pCMV-Tag2B vector (Agilent Technologies, Santa Clara, CA) at sites recognized by the restriction enzymes Eco RI and Xho I.

Techniques: Transfection, Control, Transduction, Expressing, Western Blot

Journal: Metabolism: clinical and experimental

Article Title: Endothelial senescence-associated secretory phenotype (SASP) is regulated by Makorin-1 ubiquitin E3 ligase

doi: 10.1016/j.metabol.2019.153962

Figure Lengend Snippet: (A, B) Mouse aortic endothelial cells (MAoECs) were isolated from WT and Terf2iphomo-EC-specific knockout (EKO) mice. After transduction with Ad-p90RSK-WT (+) or Ad-GFP (−) as a control for 12 h, expression of indicated proteins was analyzed by Western blotting using specific antibodies (A). Graphs represent densitometry data from immunoblots for selected proteins (B). Data represent mean ± SD (n = 3). **p <0.01. All data were analyzed by one-way ANOVA followed by the Bonferroni post hoc test. (C) D-flow–induced p90RSK activation simultaneously induces EC senescence and activation via TERF2IP S205 phosphorylation. TERF2IP S205 phosphorylation induces nuclear export of the TERF2IP-TRF2 complex, leading to senescence induced by telomere shortening/dysfunction and EC activation (i.e., NF-κB activation) induced by TERF2IP-IkB kinase binding in the cytosol. Telomere dysfunction or dislocation of the TERF2IP-TRF2 complex from telomeres precipitates trans-repression of the distinct reciprocal gene set, including MKRN1, enhancing EC senescence and activation under d-flow conditions.

Article Snippet: Plasmids containing rat wild type (WT) p90RSK1 (WT- p90rsk ) (Genebank NM031107) was generated as we previously described[ 19 ]. pLPC-human TERF2IP full length (# 12542 ) was from Addgene. pCMV-Flag-TERF2IP-WT was obtained by subcloning TERF2IP from pLPC-human TERF2IP full length into the pCMV-Tag2B vector (Agilent Technologies, Santa Clara, CA) at sites recognized by the restriction enzymes Eco RI and Xho I.

Techniques: Isolation, Knock-Out, Transduction, Control, Expressing, Western Blot, Activation Assay, Phospho-proteomics, Binding Assay

Journal: Frontiers in Genetics

Article Title: DNA damage accumulation and TRF2 degradation in atypical Werner syndrome fibroblasts with LMNA mutations

doi: 10.3389/fgene.2013.00129

Figure Lengend Snippet: Expression of shelterin subunits in LMNA mutant primary fibroblasts. (A) Western analysis of TRF2, TRF1, POT1, TIN2, hRap1, and TPP1 in control, LMNA and WRN mutant primary fibroblasts. Protein levels were normalized to β-Actin. Autoradiographies with exposures beyond the linear range are shown for clarification of bands. (B) Densitometric scanning of band intensities obtained from western analysis. Data are means ± s.d. from three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001. (C) Analysis of relative telomere lengths expressed as average telomere intensities ± s.d. in indicated cell types of control, LMNA mutant and WRN mutant fibroblasts. The signal intensities were analyzed by Q-FISH using a telomere-specific peptide nucleic acid (PNA) probe. A centromere PNA probe was used as an internal standard. Approximately 70 cells were analyzed for each cell line. The relative mean telomere lengths (±s.d.) in fibroblasts were measured by quantitative PCR. (D) TRF2 mRNA expression in normal, LMNA mutant and WRN mutant fibroblasts. TRF2 mRNA expression was detected by qRT-PCR. GAPDH mRNA expression was used as an internal standard. Data are presented as mean ± s.d. from three independent experiments. Control (82-6) values were taken as onefold.

Article Snippet: The membranes were incubated with anti-TRF2 (IMG-124A, clone 4a794.15, Imgenex, San Diego, CA, USA and sc-32106, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-TRF1 (sc-5596, Santa Cruz Biotechnology Inc), anti-POT1 (sc-27952, Santa Cruz Biotechnology Inc.), anti-TIN2 (IMG-282, clone 59B388.1, Imgenex), anti-hRap1 (IMG-289, Imgenex), anti-TPP1 (ab54685, Abcam Inc., Cambridge, MA, USA) at a dilution of 1:2000 or anti-β-actin (clone AC-15, Sigma, St Louis, MO, USA) at a dilution of 1:40,000 in a milk blocking solution, then with corresponding biotinylated secondary antibodies against mouse, rabbit or goat IgG (Vector Laboratories, Burlingame, CA, USA).

Techniques: Expressing, Mutagenesis, Western Blot, Control, Clarification Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: Frontiers in Genetics

Article Title: DNA damage accumulation and TRF2 degradation in atypical Werner syndrome fibroblasts with LMNA mutations

doi: 10.3389/fgene.2013.00129

Figure Lengend Snippet: Mean values of relative protein levels of shelterin components in primary and hTERT-immortalized fibroblasts of LMNA mutant cell lines. Protein levels in mutant lines were normalized to the average in controls, 82-6 and 82-6 hTERT.

Article Snippet: The membranes were incubated with anti-TRF2 (IMG-124A, clone 4a794.15, Imgenex, San Diego, CA, USA and sc-32106, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-TRF1 (sc-5596, Santa Cruz Biotechnology Inc), anti-POT1 (sc-27952, Santa Cruz Biotechnology Inc.), anti-TIN2 (IMG-282, clone 59B388.1, Imgenex), anti-hRap1 (IMG-289, Imgenex), anti-TPP1 (ab54685, Abcam Inc., Cambridge, MA, USA) at a dilution of 1:2000 or anti-β-actin (clone AC-15, Sigma, St Louis, MO, USA) at a dilution of 1:40,000 in a milk blocking solution, then with corresponding biotinylated secondary antibodies against mouse, rabbit or goat IgG (Vector Laboratories, Burlingame, CA, USA).

Techniques: Mutagenesis

Journal: Frontiers in Genetics

Article Title: DNA damage accumulation and TRF2 degradation in atypical Werner syndrome fibroblasts with LMNA mutations

doi: 10.3389/fgene.2013.00129

Figure Lengend Snippet: Expression of shelterin subunits in hTERT-immortalized cell lines of control, LMNA mutant and WRN mutant fibroblasts. (A) Western analysis of TRF2, TRF1, POT1, TIN2, hRap1, and TPP1 in hTERT-immortalized fibroblasts of indicated cell lines. Autoradiographies with higher exposures are shown for clarification of bands. (B) Analysis of relative telomere lengths expressed as average telomere intensities ± s.d. in indicated cell types, as shown in Figure . A minimum of 70 cells were analyzed for each cell lines. Relative mean telomere lengths (±s.d.) were measured in hTERT-immortalized fibroblasts using qPCR. (C) TRF2 mRNA expression in hTERT-immortalized normal, LMNA mutant and WRN mutant fibroblasts. TRF2 mRNA expression was detected by qRT-PCR. GAPDH mRNA was used as an internal standard. Data are presented as mean ± s.d. from three independent experiments. The control (82-6) value was taken as onefold. (D) Expression of TRF2 protein after proteasome inhibition in LMNA mutant cells. hTERT-immortalized cell lines of control and LMNA mutant fibroblasts were incubated in the presence or absence of MG-132 for 6 h and TRF2 protein levels were detected using immunoblotting.

Article Snippet: The membranes were incubated with anti-TRF2 (IMG-124A, clone 4a794.15, Imgenex, San Diego, CA, USA and sc-32106, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), anti-TRF1 (sc-5596, Santa Cruz Biotechnology Inc), anti-POT1 (sc-27952, Santa Cruz Biotechnology Inc.), anti-TIN2 (IMG-282, clone 59B388.1, Imgenex), anti-hRap1 (IMG-289, Imgenex), anti-TPP1 (ab54685, Abcam Inc., Cambridge, MA, USA) at a dilution of 1:2000 or anti-β-actin (clone AC-15, Sigma, St Louis, MO, USA) at a dilution of 1:40,000 in a milk blocking solution, then with corresponding biotinylated secondary antibodies against mouse, rabbit or goat IgG (Vector Laboratories, Burlingame, CA, USA).

Techniques: Expressing, Control, Mutagenesis, Western Blot, Clarification Assay, Quantitative RT-PCR, Inhibition, Incubation