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