Journal: Cell proliferation

Article Title: Super-Enhancer Target Gene CBP/p300-Interacting Transactivator With Glu/Asp-Rich C-Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling.

doi: 10.1111/cpr.13817

Figure Lengend Snippet: FIGURE 3 | Overexpression of CITED2 at the animal level is able to alleviate PH. (A) The results of gene enrichment analysis in normoxia and hypoxia showed that CITED2 was downregulated in hypoxia, showing a negative correlation. (B–D) Expression of CITED2 mRNA and protein in the pulmonary arteries of model mice. (E) Schematic diagram of lentivirus model mouse construction. (F) The decreased cardiopulmonary function of model mice was effectively reversed by overexpression of CITED2. (G) Right ventricular systolic blood pressure and right ventricular specific gravity were improved in the CITED2 overexpression group. (H, I) H&E and Masson staining of tissue sections of mouse pulmonary arterioles. (K) The ex- pression of PCNA was downregulated in the pulmonary arteries of mice in the lentivirus-treated group. (J) Tissue immunofluorescence experiments showed that the expression of Ki67 in small pulmonary vessels decreased significantly after CITED2 overexpression. A: artery. (L, M) The expression of cycle-related proteins in the pulmonary arteries of mice in the lentivirus-treated group was downregulated. LV: lentivirus. (Bar = mean ± S.E.M, **p < 0.01; ***p < 0.001).

Article Snippet: After the cells of the same model were fixed with paraformaldehyde (Tissue sections need dewaxing first), they were stained with CITED2 probe (BOSTER, Product number MK3868- m), and the distribution characteristics of cytoplasm and nucleus and subcellular localisation of CITED2 were determined.

Techniques: Over Expression, Expressing, Staining, Immunofluorescence

Journal: Cell proliferation

Article Title: Super-Enhancer Target Gene CBP/p300-Interacting Transactivator With Glu/Asp-Rich C-Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling.

doi: 10.1111/cpr.13817

Figure Lengend Snippet: FIGURE 4 | Overexpression of CITED2 can affect the proliferation of smooth muscle cells and hinder cell cycle progression. (A) Western blot re- sults of efficiency detection after overexpression of CITED2. (B) The results of the CCK8 experiment indicated that overexpression of CITED2 could affect the growth and viability of mPASMCs. (C) Overexpression of CITED2 affected the expression of PCNA in cells. (D) After overexpression of CITED2, the proportion of cells in the G2/M and S phase was reduced, which affected cell division. (E, F) The results of the EdU cell fluorescence assay and Ki67 cell fluorescence assay indicated that overexpression of CITED2 could reduce cell proliferation. Red and green represent remark- ably proliferating cells. (G, H) Overexpression of CITED2 at the cellular level can affect the expression levels of multiple proteins in the cell cycle. (Bar = mean ± S.E.M, **p < 0.01; ***p < 0.001).

Article Snippet: After the cells of the same model were fixed with paraformaldehyde (Tissue sections need dewaxing first), they were stained with CITED2 probe (BOSTER, Product number MK3868- m), and the distribution characteristics of cytoplasm and nucleus and subcellular localisation of CITED2 were determined.

Techniques: Over Expression, Western Blot, Expressing, Fluorescence

Journal: Cell proliferation

Article Title: Super-Enhancer Target Gene CBP/p300-Interacting Transactivator With Glu/Asp-Rich C-Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling.

doi: 10.1111/cpr.13817

Figure Lengend Snippet: FIGURE 6 | FOXJ3 cooperates with SEs to regulate the transcription of CITED2. (A) Schematic representation of overexpression plasmid con- struction and transfection. (B–D) In the dual-luciferase experiment, plasmids containing three SEs motifs were co-transfected with FOXJ3 plasmids into mPASMCs. When both were present, the luciferase activity was the greatest. (Bar = mean ± S.E.M, *p < 0.05).

Article Snippet: After the cells of the same model were fixed with paraformaldehyde (Tissue sections need dewaxing first), they were stained with CITED2 probe (BOSTER, Product number MK3868- m), and the distribution characteristics of cytoplasm and nucleus and subcellular localisation of CITED2 were determined.

Techniques: Over Expression, Plasmid Preparation, Transfection, Luciferase, Activity Assay

Journal: Cell proliferation

Article Title: Super-Enhancer Target Gene CBP/p300-Interacting Transactivator With Glu/Asp-Rich C-Terminal Domain, 2 Cooperates With Transcription Factor Forkhead Box J3 to Inhibit Pulmonary Vascular Remodeling.

doi: 10.1111/cpr.13817

Figure Lengend Snippet: FIGURE 9 | Differential effects of CITED2 on PASMC proliferation under normoxia and hypoxia. Normally, the H3K27ac modification of the SEs and promoter regions of CITED2 is significant. Downstream proteins influenced by CITED2, such as cyclin proteins, CDKs and PCNA, decreased. SMC proliferation is also diminished. However, the phenomenon is reversed under hypoxia.

Article Snippet: After the cells of the same model were fixed with paraformaldehyde (Tissue sections need dewaxing first), they were stained with CITED2 probe (BOSTER, Product number MK3868- m), and the distribution characteristics of cytoplasm and nucleus and subcellular localisation of CITED2 were determined.

Techniques: Modification

Journal: International Journal of Molecular Sciences

Article Title: Acetyltransferase P300 Regulates Glucose Metabolic Reprogramming through Catalyzing Succinylation in Lung Cancer

doi: 10.3390/ijms25021057

Figure Lengend Snippet: p300 catalyzes lysine succinylation in LUAD cells. ( A , B ) EP300 deficiency impairs Kac and Ksucc in both A549 and H1975 cells. Kac and Ksucc levels in WT and EP300 KO cells were determined by Western blot with a pan-antibody, respectively. ( C ) Ksucc levels of A549 and H1975 cells were inhibited by A485 in a dose-dependent manner. A549 and H1975 cells were incubated with A485 in increasing concentration. Ksucc levels were determined by Western blot with a pan-anti-succinyllysine antibody. ( D ) Sodium succinate enhanced Ksucc levels in A549 and H1975 cells in a dose-dependent manner. The levels of Ksucc were assessed by using a pan-anti-succinyllysine antibody in a Western blot after a culture procedure with sodium succinate. ( E ) p300-catalyzed succinylation was decreased by A485 and was enhanced by sodium succinate. WT and EP300 KO cells (A549 and H1975) were treated with or without A485 and sodium succinate. Lysates of the cells above were analyzed by Western blot with a pan-anti-succinyllysine antibody.

Article Snippet: Mouse monoclonal anti-p300 antibody , Santa Cruz, Santa Cruz, CA, USA , sc-48343.

Techniques: Western Blot, Incubation, Concentration Assay

Journal: International Journal of Molecular Sciences

Article Title: Acetyltransferase P300 Regulates Glucose Metabolic Reprogramming through Catalyzing Succinylation in Lung Cancer

doi: 10.3390/ijms25021057

Figure Lengend Snippet: Deletion of EP300 decreases Ksucc levels on various proteins. ( A ) Schematic representation of the experimental workflow for the quantitative succinylproteomics analysis. ( B ) The intensities of Ksucc sites and succinylated peptides with or without the loss of EP300 are compared and shown in the scatter diagram. ( C ) Volcano plot showed that up-regulated and down-regulated Ksucc sites of LUAD cells in response to the deletion of EP300 ( p -value < 0.05). ( D ) The bar chart displayed the sites and proteins with differences in the difference analysis. ( E ) p300-mediated succinylation mainly occurred in the cytosol (41.53%) and mitochondria (38.02%) of LUAD cells. WolF Psort software ( https://www.psort.org/ , NAKAI Lab, Tokyo, Japan) was used to annotate the subcellular localization of proteins. ( F ) The pie chart indicated that more than 99% of Ksucc’s substrates were not histones. ( G ) The frequency of amino acids occurring near the Ksucc site is shown by the iceLogo. The size or color intensity of an amino acid reflects the difference in the frequency of the amino acid.

Article Snippet: Mouse monoclonal anti-p300 antibody , Santa Cruz, Santa Cruz, CA, USA , sc-48343.

Techniques: Software

Journal: International Journal of Molecular Sciences

Article Title: Acetyltransferase P300 Regulates Glucose Metabolic Reprogramming through Catalyzing Succinylation in Lung Cancer

doi: 10.3390/ijms25021057

Figure Lengend Snippet: p300-mediated Ksucc modulates tumor carbohydrate metabolism. ( A ) p300-dependent Ksucc sites enriched in the metabolic process and protein binding. GO enrichment was conducted on the differential succinylation sites identified in B,C. ( B ) p300-related Ksucc mainly targets glycolysis/gluconeogenesis in LUAD cells. KEGG pathway analysis proceeded based on the differential analysis of Ksucc sites. ( C ) The majority of succinylated proteins were classified into groups related to energy metabolism. Function categorization was performed by the KOGs database. ( D ) p300-mediated Ksucc specifically targets glycolytic enzymes. The interaction network is from the STRING database, and the size of the node positively corresponds to the degrees provided by STRING.

Article Snippet: Mouse monoclonal anti-p300 antibody , Santa Cruz, Santa Cruz, CA, USA , sc-48343.

Techniques: Protein Binding

Journal: International Journal of Molecular Sciences

Article Title: Acetyltransferase P300 Regulates Glucose Metabolic Reprogramming through Catalyzing Succinylation in Lung Cancer

doi: 10.3390/ijms25021057

Figure Lengend Snippet: p300 succinylates key glycolytic enzymes. ( A ) The succinylated proteomes were subjected to a hierarchical clustering analysis and separated into four portions by p -value in the differential analysis, designated Q1 to Q4. ( B , C ) For each Q category, KEGG pathway ( B ) and GO categorization ( C ) enrichment were carried out (* p < 0.05, ** p < 0.01, *** p < 0.001). ( D ) Top five succinylated glycolytic enzymes catalyzed by p300. Shown are the top five Ksucc sites on glycolytic enzymes selected by p -value in differential analysis. ( E – I ) Mass spectrometry evidence of five succinylated sites (PCK2-K108succ, PGK1 -K323succ, PFKL-K677succ, PKM -K166succ, and ALDOA -K13succ). ( J , K ) The bars showed the expression levels of the three subunits of PFK (PFKM, PFKP, PFKL) in normal lung tissues ( n = 58, *** p < 0.001, values are expressed as mean ± SEM) and tumors. ( L ) Every succinyl modification site in the Q2 category in A. ( M ) The top 10 proteins of glycolysis in the center of the PPI interaction network in D. ( N ) The figure shows the overlap of succinylated proteins of the three ranges mentioned in D,L,M (The green circle refers to the Ksucc sites in L; The orange circle refers to the 5 Ksucc sites in D; The blue circle refers to the 10 proteins in M; The numbers in the colored areas represent the number of overlapping proteins).

Article Snippet: Mouse monoclonal anti-p300 antibody , Santa Cruz, Santa Cruz, CA, USA , sc-48343.

Techniques: Mass Spectrometry, Expressing, Modification

Journal: International Journal of Molecular Sciences

Article Title: Acetyltransferase P300 Regulates Glucose Metabolic Reprogramming through Catalyzing Succinylation in Lung Cancer

doi: 10.3390/ijms25021057

Figure Lengend Snippet: p300-mediated PGK1 -K323succ enhances tumor glycolysis. ( A , B ) The Ksucc of PGK1 and ALDOA were detected by IP assays and immunoblotting in cells (A549 or H1975) with indicated antibodies. ( C – E ) The glucose uptake, LDH activity, and lactic acid production of PGK1 -WT and ALDOA -WT or PGK1 -K323R and ALDOA -K13R LUAD cells were detected by indicated kits ( n = 5–6, *** p < 0.001, values are expressed as mean ± SEM). ( F ) The seahorse analysis showed that PGK1 -K323R reduces the extracellular acidification of LUAD cells compared to PGK1 -WT. ( G ) The bar plot showed the relative glycolysis, glycolytic capacity, and glycolytic reserve of PGK1 -WT and PGK1 -K323R in seahorse results ( n = 5, ** p < 0.01, *** p < 0.001, values are expressed as mean ± SEM). ( H ) The seahorse analysis showed that ALDOA -K13R cannot reduce the extracellular acidification of LUAD cells compared to ALDOA -WT. ( I ) The bar plot showed the relative glycolysis, glycolytic capacity, and glycolytic reserve of ALDOA -WT and ALDOA -K13R in seahorse results ( n = 5, ns, no significance, values are expressed as mean ± SEM).

Article Snippet: Mouse monoclonal anti-p300 antibody , Santa Cruz, Santa Cruz, CA, USA , sc-48343.

Techniques: Western Blot, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Acetyltransferase P300 Regulates Glucose Metabolic Reprogramming through Catalyzing Succinylation in Lung Cancer

doi: 10.3390/ijms25021057

Figure Lengend Snippet: EP300 deficiency impairs glycolysis and malignant phenotypes of LUAD cells. ( A , B ) EP300 deficiency of A549 and H1975 cells reduces extracellular acidification. The bar charts represent glycolysis, glycolytic capacity, and glycolytic reserve, respectively ( n = 4–6, ns, no significance, * p < 0.05, *** p < 0.001, values are expressed as mean ± SEM). ( C , D ) EP300 deficiency of A549 and H1975 cells reduces the glucose uptake and increases the extracellular glucose concentrations ( n = 4–5, * p < 0.05, *** p < 0.001, values are expressed as mean ± SEM). ( E , F ) EP300 deficiency of A549 and H1975 cells reduces the LDH activity and lactic acid production ( n = 5–6, *** p < 0.001, values are expressed as mean ± SEM). ( G ) Adding the inhibitor of p300 reduces the extracellular acidification as well ( n = 5, * p < 0.05, ** p < 0.01, *** p < 0.001, values are expressed as mean ± SEM). ( H ) Adding the inhibitor of p300 reduces the lactic acid production as expected ( n = 7, *** p < 0.001, values are expressed as mean ± SEM). ( I , J ) Adding the inhibitor of p300 reduces the Lactic acid production with or without treatment of glutamine or glucose. Cells incubated in a medium without glucose and glutamine were treated with extra glutamine or glucose in the presence of A485 ( n = 5, *** p < 0.001, values are expressed as mean ± SEM). ( K – N ) EP300 deficiency suppresses the proliferation ( A ), stemness ( B ), migration ( C ), and invasion ( D ) of LUAD cells. ( K ). Cells were plated in a complete medium and switched to the indicated medium at 0 h ( n = 5, *** p < 0.001, values are expressed as mean ± SEM). ( L ) Cells were plated in a complete medium and dyed with crystal violet. The magnification of the microscope is ×100. The number of formed colonies was provided by ImageJ 1.51 software. ( n = 3, ** p < 0.01, values are expressed as mean ± SEM). ( M ) Cells were completely attached to the wall and plated in a complete medium. Migration rates were provided by ImageJ 1.51 software. ( n = 3, ** p < 0.01, values are expressed as mean ± SEM). ( N ) After 48 h of incubation in the Transwell chamber, cells were fixed with glutaraldehyde and stained with crystal violet. Cells in the lower chamber were analyzed by ImageJ 1.51 software. ( n = 3, ** p < 0.01, values are expressed as mean ± SEM).

Article Snippet: Mouse monoclonal anti-p300 antibody , Santa Cruz, Santa Cruz, CA, USA , sc-48343.

Techniques: Activity Assay, Incubation, Migration, Microscopy, Software, Staining

Journal: International Journal of Molecular Sciences

Article Title: Acetyltransferase P300 Regulates Glucose Metabolic Reprogramming through Catalyzing Succinylation in Lung Cancer

doi: 10.3390/ijms25021057

Figure Lengend Snippet: EP300 coincides with advanced tumor stages and poor prognosis of LUAD individuals. ( A ) The expression of EP300 in tumors was higher than that in adjacent tissues. The expression of EP300 in tumors and adjacent tissues was determined by IHC staining; scale bar, 29.34 μm. ( B ) The scatter bar plot shows the IHC scores of overall p300 expression, and the paired t -test was used (n of pairs = 125, *** p < 0.001). ( C ) The stacked bar plot displays the proportion of p300-positive individuals in tumors and adjacent tissues. ( D ) LUAD patients with p300 cytoplasmic localization positivity accounted for 89.35% ( n = 169). ( E , F ) The bar chart with scatter points suggests that p300 is mainly located in the cytoplasm in tumors but in the nucleus in adjacent lung tissues (Tumor, n = 169; Adjacent, n = 93, ** p < 0.01). ( G ) In LUAD cells, p300 is mainly located in the cytoplasm ( n = 169, *** p < 0.001). ( H ) OS (overall survival) rates of all individuals with high or low EP300 expression levels were determined by Kaplan–Meier analysis ( n = 143). ( I ) Kaplan–Meier analysis of patients with early-stage LUAD. AJCC stages I and II were identified as early stages ( n = 90). ( J ) Kaplan–Meier analysis of patients with late-stage LUAD. AJCC stages III and IV were identified as late-stage, and one individual lost the staging information ( n = 52). ( K , L ) Univariate and multivariate COX regression analyses of EP300 and other clinicopathological variables ( n = 143, * p < 0.05, values are expressed as mean ± SEM).

Article Snippet: Mouse monoclonal anti-p300 antibody , Santa Cruz, Santa Cruz, CA, USA , sc-48343.

Techniques: Expressing, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: Acetyltransferase P300 Regulates Glucose Metabolic Reprogramming through Catalyzing Succinylation in Lung Cancer

doi: 10.3390/ijms25021057

Figure Lengend Snippet: The reagents or resources involved in this study.

Article Snippet: Mouse monoclonal anti-p300 antibody , Santa Cruz, Santa Cruz, CA, USA , sc-48343.

Techniques: Recombinant, Immunoprecipitation, Bicinchoninic Acid Protein Assay, Glucose Assay, Lactate Dehydrogenase Assay, Acid Assay, CRISPR, Plasmid Preparation, Microarray, Staining, Software

Journal: eLife

Article Title: SIRT2 deacetylase regulates the activity of GSK3 isoforms independent of inhibitory phosphorylation

doi: 10.7554/eLife.32952

Figure Lengend Snippet: ( A ) Scatter plot showing cardiac hypertrophy, as measured by Heart weight/Tibia Length (HW/TL) ratio of 8 weeks old 129/Sv mice treated with either vehicle or isoproterenol (ISO) at the dose of 10 mg/kg/day. ISO was continuously infused for 7 days using osmotic mini-pumps. n = 9–10 mice per group. Data is presented as mean ± s.d, *p<0.05. Student’s t test was used to calculate the p values. ( B ) Scatter plot representing left ventricular posterior wall thickness of 8 weeks old 129/Sv mice treated with either vehicle or ISO at the dose of 10 mg/kg/day. ISO was continuously infused for 7 days using osmotic mini-pumps. n = 6 mice per group. Data is presented as mean ± s.d, *p<0.05. Student’s t test was used to calculate the p values. ( C ) Scatter plot indicating the contractile functions of heart as represented by ejection fraction of 8 weeks old 129/Sv mice treated with either vehicle or ISO at the dose of 10 mg/kg/day. ISO was continuously infused for 7 days using osmotic mini-pumps. n = 6 mice per group. Data is presented as mean ± s.d, *p<0.05. Student’s t test was used to calculate the p values. ( D ) Histogram showing GSK3β activity assay in heart lysates of vehicle or ISO-treated 8 weeks old 129/Sv mice. Mice were treated with either vehicle or ISO at the dose of 10 mg/kg/day for 7 days using osmotic mini-pumps. GSK3β was immunoprecipitated from the heart lysates of vehicle or ISO infused mice using anti-GSK3β antibody, clone GSK-4B (Sigma). The immunoprecipitated GSK3β was incubated with the peptide substrate in the presence of γ− 32 P-ATP. The incorporation of 32 P into the GSK3β peptide substrate, which contains specific phosphorylation residues of GSK3β was measured. n = 10 mice per group. Data is presented as mean ± s.d, *p<0.05. Student’s t test was used to calculate the p values. ( E ) Eight weeks old 129/Sv mice were treated with either vehicle or ISO at the dose of 10 mg/kg/day for 7 days using osmotic mini-pumps. GSK3β was immunoprecipitated from the heart lysates of vehicle or ISO infused mice using anti-GSK3β antibody (sc-9166, Santa Cruz Biotechnolgy) and the affinity resin immobilized with protein A/G. Western blotting analysis was performed to detect the levels of GSK3β acetylation (Ac-Lys) by anti-acetyl-lysine antibody. IgG was used as negative control in this assay. Heart tissue lysates (WCL) were probed for indicated proteins by western blotting. ANP was used as a positive control to assess cardiac hypertrophy in ISO infused mice. n = 4 mice per group. # marked western blotting images denotes SIRT2 antibody (#12650; Cell Signaling), used in this assay detects single band. ( F ) Histogram showing relative acetylated GSK3β in vehicle and ISO-treated mice heart tissues, as measured from . Signal intensities of acetylated GSK3β and GSK3β were measured by densitometry analysis (ImageJ software). n = 4 mice per group. Data is presented as mean ± s.d. *p<0.05. Student’s t test was used to calculate the p values. ( G ) GSK3β was immunoprecipitated from heart tissues of 8 weeks old 129/Sv mice using anti-GSK3β antibody (sc-9166, Santa Cruz Biotechnology), and the affinity resin with protein A/G immobilized. Western blotting was performed to detect GSK3β interaction with p300 using anti-p300 antibody. IgG was used as a negative control. Whole cell lysates (WCL) were probed for the presence of GSK3β and p300 by western blotting. ( H ) Co-localization of GSK3β with p300 was assessed in 293 T cells by confocal microscopy. The antibodies used are anti-GSK3β (sc-9166, Santacruz), and p300 (05–257, Millipore). DAPI was used to stain the nucleus. Expanded images (right small boxes) show yellow color in the merge image, indicating the co-localization of GSK3β (Green) and p300 (Red) in the nucleus. ( I ) In vitro binding assay to test the direct interaction between GSK3β and p300. Recombinant p300 (Millipore # 2273152) was incubated with recombinant GST or GST-GSK3β, purified from E. coli BL21 (DE3) by affinity chromatography using Glutathione Sepharose 4B. ( J ) Western blotting analysis showing the acetylation and activity of GSK3β in rat neonatal cardiomyocytes infected with adenovirus expressing either luciferase shRNA (control) or p300 shRNA (p300-KD) for 72 hr. Depletion of p300 was confirmed by western blotting. GSK3β was immunoprecipitated from control and p300-KD cells using anti-GSK3β antibody (sc-9166, Santa Cruz Biotechnology) and the affinity resin immobilized with protein A/G. Western blotting was performed to detect acetylation of GSK3β using the anti Ac-Lysine antibody. GSK3β activity was measured by assessing the phosphorylation of glycogen synthase (p–GS). Site-specific antibodies were used to detect the phosphorylation of GSK3β at indicated residues in cardiomyocyte lysates (WCL). ( K ) Histogram showing the quantification of relative acetylated GSK3β in control and p300 depleted (p300-KD) rat neonatal cardiomyocytes, as measured from . Rat neonatal cardiomyocytes were infected with adenovirus expressing either luciferase shRNA (control) or p300 shRNA (p300-KD) for 72 hr. Signal intensities of acetylated GSK3β and GSK3β were quantified by densitometry analysis (ImageJ software). n = 3 independent experiments. Data is presented as mean ± s.d. *p<0.05. Student’s t test was used to calculate the p values. ( L ) Histogram depicting the activity of GSK3β in control and p300 depleted (p300-KD) rat neonatal cardiomyocytes, as measured by the ratio of phosphorylation of glycogen synthase vs total glycogen synthase from . Rat neonatal cardiomyocytes were infected with adenovirus expressing either luciferase shRNA (control) or p300 shRNA (p300-KD) for 72 hr. Signal intensities of phospho-glycogen synthase and glycogen synthase were measured by densitometry analysis (ImageJ software). n = 3 independent experiments. Data is presented as mean ± s.d. *p<0.05. Student’s t test was used to calculate the p values. ( M ) Western blotting analysis showing the acetylation of GSK3β in rat neonatal cardiomyocytes infected with either control (Ad-null) or p300 overexpressing adenovirus (Ad-p300) for 24 hr. Overexpression of p300 was confirmed by western blotting. GSK3β was immunoprecipitated using anti-GSK3β antibody (sc-9166, Santacruz) and the affinity resin with protein A/G immobilized. Site-specific antibodies were used to detect the phosphorylation of GSK3β at indicated residues in cell lysates (WCL). ( N ) Western blotting analysis showing the activity of GSK3β in rat neonatal cardiomyocytes infected with control (Ad-null) or p300 expressing adenovirus (Ad-p300) for 24 hr. Overexpression of p300 was confirmed by western blotting and the activity of GSK3β was probed by assessing the levels of p-GS and GS by western blotting. ( O ) Histogram showing the activity of GSK3β in control (Ad-Null) or p300 overexpressing (Ad-p300) rat neonatal cardiomyocytes, as measured by the ratio of phosphorylation of glycogen synthase vs total glycogen synthase from . Signal intensities of phospho-glycogen synthase and glycogen synthase were assessed by densitometry analysis (ImageJ software). n = 3 independent experiments. Data is presented as mean ± s.d. *p<0.05. Student’s t test was used to calculate the p values. ( P ) In vitro kinase assay showing the activity of acetylated and non-acetylated GSK3β. Human GSK3β with HA tag was overexpressed in HeLa cells by transfection of the plasmid pcDNA3-HA-GSK3β. HA-GSK3β was immunoprecipitated using HA-coupled agarose beads (Sigma-Aldrich) and the HA-GSK3β was acetylated by recombinant p300 (Millipore), in the presence or absence of Acetyl-CoA (Ac-CoA) in HAT buffer. The enzymatic activity of GSK3β was measured against glycogen synthase (GS)-peptide. n = 6 independent experiments. Data is presented as mean ± s.d. *p<0.05. One-way ANOVA was used to calculate the p values.

Article Snippet: Recombinant protein , p300 , Merck Millipore , 14–418 , http://dx.doi.org/10.1038/s41418-018-0069-8.

Techniques: Activity Assay, Immunoprecipitation, Incubation, Phospho-proteomics, Western Blot, Negative Control, Positive Control, Software, Confocal Microscopy, Staining, In Vitro, Binding Assay, Recombinant, Purification, Affinity Chromatography, Infection, Expressing, Luciferase, shRNA, Control, Over Expression, Kinase Assay, Transfection, Plasmid Preparation

Journal: eLife

Article Title: SIRT2 deacetylase regulates the activity of GSK3 isoforms independent of inhibitory phosphorylation

doi: 10.7554/eLife.32952

Figure Lengend Snippet: ( A ) Western blot analysis of acetylated GSK3β in heart samples of 9 months old WT and SIRT2-KO littermates. GSK3β was immunoprecipitated from heart tissue lysates of WT and SIRT2-KO mice using anti-GSK3β antibody (sc-9166, Santa Cruz Biotechnolgy), and the affinity resin immobilized with protein A/G. Western blotting was performed to detect GSK3β acetylation by anti-Ac-Lysine antibody. IgG was used as a negative control. Whole cell lysates (WCL) were probed for the SIRT2 and GAPDH by western blotting. n = 4 mice per group. ( B ) Histogram showing relative acetylated GSK3β in 9 months old WT and SIRT2-KO mice heart tissues, as measured from . Signal intensities of acetylated GSK3β and GSK3β were measured by densitometry analysis (ImageJ software). n = 4 mice per group. Data is presented as mean ± s.d, *p<0.05. Student’s t test was used to calculate the p values. ( C ) GSK3β was immunoprecipitated from heart tissue lysates of 8 weeks old 129/Sv mice using anti-GSK3β antibody (sc-9166, Santa Cruz Biotechnolgy ), and the affinity resin immobilized with protein A/G. GSK3β interaction with SIRT2 was tested by western blotting using anti-SIRT2 antibody. IgG was used as negative control. Heart lysates was probed for indicated proteins by western blotting. ( D ) In vitro binding assay to test the interaction between GSK3β and SIRT2. Flag-SIRT2 was overexpressed in 293 cells by a plasmid encoding human Flag-SIRT2. Recombinant His or His-GSK3β was purified from E. coli BL21 (DE3) by Ni-NTA affinity chromatography and were incubated with 293 T cell lysates overexpressing human Flag-SIRT2. Interaction between GSK3β and SIRT2 was tested by western blotting. # marked western images denotes SIRT2 antibody used in this assay detects single band. ( E ) In vitro deacetylation assay showing SIRT2 as GSK3β deacetylase. Human HA-GSK3β was overexpressed in HeLa cells by transfection of the plasmid pcDNA3-HA-GSK3β. HA-GSK3β was immunoprecipitated using HA-coupled agarose beads (Sigma-Aldrich) and the HA-GSK3β was acetylated by recombinant p300 (Millipore), in the presence or absence of Acetyl-CoA (Ac-CoA) in HAT buffer. The acetylated HA-GSK3β was further incubated with either Flag-tagged SIRT2 or SIRT2-H187Y, which were immunoprecipitated from HEK 293 cell lysates overexpressing respective plasmids encoding Flag-tagged WT or SIRT2-H187Y using agarose beads conjugated to anti-Flag antibody (Sigma A2220). The deacetylation reaction was carried out in the presence or absence of NAD + in a HDAC buffer. GSK3β acetylation was analyzed by western blotting using anti-Ac-Lysine antibody. # marked western images denotes SIRT2 antibody used in this assay detects single band. ( F ) In vitro kinase assay depicting the activity of acetylated and deacetylated GSK3β. Human HA-GSK3β was overexpressed in HeLa cells by transfection of the plasmid pcDNA3-HA-GSK3β. Recombinant HA-GSK3β was immunoprecipitated using HA-coupled beads and was acetylated by recombinant p300 in the presence or absence of Acetyl-CoA (Ac-CoA) in HAT buffer. Acetylated GSK3β was further deacetylated by either Flag-tagged WT or SIRT2-H187Y (SIRT2-HY), a catalytic inactive mutant of SIRT2, which was immunoprecipitated from HEK 293 cells, overexpressed with plasmid encoding Flag-tagged WT or SIRT2-H187Y using agarose beads conjugated to anti-Flag antibody (Sigma A2220). The deacetylation reaction was carried out in the presence or absence of NAD + in a HDAC buffer and further enzymatic activity of GSK3β was measured against glycogen synthase (GS)-peptide, as described in the Materials and methods section. n = 5. Data is presented as mean ± s.d. *p<0.05. One-way ANOVA was used to calculate the p values. ( G ) Western blot analysis of acetylated GSK3β from control or SIRT2-depleted (SIRT2-KD) cardiomyocytes. Neonatal rat cardiomyocytes were transfected with either non-targeting (control) or siRNA targeting SIRT2 using Lipofectamine RNAiMAX reagent for 72 hr. SIRT2 depletion was confirmed by Western blotting. Total cellular acetylation was probed by anti-Ac-Lysine antibody to test the effect of SIRT2 depletion in cardiomyocytes. GSK3β was immunoprecipitated from these cell lysates using anti-GSK3β antibody (sc-9166, Santa Cruz Biotechnolgy), and the affinity resin immobilized with protein A/G. Western blotting was performed to detect acetylation of GSK3β by anti-Ac-Lysine antibody. Cell lysates (WCL) from control and SIRT2-KD cardiomyocytes were probed for indicated proteins by western blotting. ( H ) Western blotting analysis of hearts lysates from 9 months old WT and SIRT2-KO mice littermates for indicated proteins. n = 4 mice per group. ( I ) Histogram showing activity of GSK3β in WT and SIRT2-KO mice hearts at 9 months of age. GSK3β was immunoprecipitated from the heart lysates of WT and SIRT2-KO mice using anti-GSK3β antibody, clone GSK-4B (Sigma). The immunoprecipitated GSK3β was incubated with the peptide substrate in the presence of γ− 32 P-ATP. The incorporation of 32 P into the GSK3β Peptide Substrate, which contains specific phosphorylation residue of GSK3β was measured. n = 6 mice per group. Data is presented as mean ± s.d. *p<0.05. Student’s t test was used to calculate the p values. ( J ) In vitro deacetylation assay to test whether SIRT2 deacetylates K183 residue of GSK3β. HA-tagged GSK3β or GSK3β-K183R was overexpressed in HeLa cells and was immunoprecipitated using HA-coupled beads. HA-tagged WT-GSK3β or GSK3β-K183R were incubated with Flag-SIRT2 immunoprecipitated from HEK 293 T cells using agarose beads conjugated to Anti-Flag antibody (Sigma A2220). The deacetylation reaction was carried out in the presence or absence of NAD + in a deacetylation buffer. Acetylation status of GSK3β was analyzed by western blotting. # marked western images denotes SIRT2 antibody used in this assay detects single band. ( K ) Histogram showing relative acetylation of HA-tagged GSK3β or GSK3β-K183R, which was incubated with Flag-SIRT2. The data is generated from . Signal intensities of acetylated-GSK3β and GSK3β were measured by densitometry analysis (ImageJ software). n = 4 independent experiments. Data is presented as mean ± s.d. *p<0.05. One-way ANOVA was used to calculate the p values. ( L ) Histogram showing binding of γ− 32 P-ATP to acetylated and deacetylated His-GSK3β. Recombinant His-GSK3β was purified from E. coli BL 21 (DE3) by Ni-NTA affinity chromatography. Purified His-GSK3β was acetylated by recombinant p300 in the presence of Ac-CoA in HAT buffer. Acetylated His-GSK3β was further deacetylated by Flag-SIRT2 immunoprecipitated from HEK 293 T cells. The binding of γ− 32 P-ATP to acetylated and deacetylated His-GSK3β was assessed by the protocol described in Materials and methods section. n = 4. Data is presented as mean ± s.d. *p<0.05. One-way ANOVA was used to calculate the p values. ( M ) Histogram showing activity of WT or mutants of GSK3β. HA-tagged WT or mutants of GSK3β was immunoprecipitated from HeLa cells transfected with respective plasmids using HA-coupled agarose beads. The enzymatic activity of GSK3β was measured against glycogen synthase (GS)-peptide, as described in the Materials and methods section. n = 4. Data is presented as mean ± s.d. *p<0.05. One-way ANOVA was used to calculate the p values.

Article Snippet: Recombinant protein , p300 , Merck Millipore , 14–418 , http://dx.doi.org/10.1038/s41418-018-0069-8.

Techniques: Western Blot, Immunoprecipitation, Negative Control, Software, In Vitro, Binding Assay, Plasmid Preparation, Recombinant, Purification, Affinity Chromatography, Incubation, Histone Deacetylase Assay, Transfection, Kinase Assay, Activity Assay, Mutagenesis, Control, Phospho-proteomics, Residue, Generated

Journal: eLife

Article Title: SIRT2 deacetylase regulates the activity of GSK3 isoforms independent of inhibitory phosphorylation

doi: 10.7554/eLife.32952

Figure Lengend Snippet: ( A ) Western blot analysis showing acetylation status of both isoforms of GSK3 in control or SIRT2 depleted (SIRT2-KD) cardiomyocytes. Neonatal rat cardiomyocytes were transfected with either non-targeting or siRNA pool targeting SIRT2 using Lipofectamine RNAiMAX reagent for 72 hr. SIRT2 depletion was confirmed by western blotting. GSK3 was immunoprecipitated from cell lysates using anti-GSK3 antibody and the affinity resin immobilized with protein A/G. Western blotting was performed to detect GSK3α/β acetylation by anti-Ac-Lysine antibody. Cell lysates was probed for SIRT2 and actin antibodies by western blotting. ( B ) Histogram showing relative acetylated-GSK3α and GSK3β in control and SIRT2-depleted (SIRT2-KD) cardiomyocytes, as measured from . Signal intensities of acetylated-GSK3α and acetylated-GSK3β were measured by densitometry analysis (ImageJ software). n = 3. Data is presented as mean ± s.d. *p<0.05. Student’s t test was used to calculate the p values. ( C ) Histogram showing enzymatic activity of acetylated and deacetylated GSK3α. Recombinant HA- GSK3α was immunoprecipitated from HeLa cells overexpressing pcDNA-HA-GSK3α using HA-coupled agarose beads. Immunoprecipitated HA-GSK3α was acetylated by p300 in the presence of Acetyl-CoA (Ac-CoA) in HAT buffer. Acetylated GSK3α was further deacetylated by Flag-SIRT2 immunoprecipitated from HEK 293 T cells overexpressing plasmid encoding Flag-tagged SIRT2-WT using agarose beads conjugated to anti-Flag antibody (Sigma A2220). The enzymatic activity of GSK3α was measured against glycogen synthase (GS)-peptide. n = 5. Data is presented as mean ± s.d. *p<0.05. One-way ANOVA was used to calculate the p values. ( D ) Annotation of representative tandem mass spectra of trypsin-digested GSK3α, depicting K99, K246 acetylation. ( E ) Protein sequence alignment of the modeled region of GSK3α and the structure of GSK3β. ( F ) Cartoon, surface representation of the homology model of GSK3α (highlighted is the adenine nucleotide-binding pocket and position of K246 residue).

Article Snippet: Recombinant protein , p300 , Merck Millipore , 14–418 , http://dx.doi.org/10.1038/s41418-018-0069-8.

Techniques: Western Blot, Control, Transfection, Immunoprecipitation, Software, Activity Assay, Recombinant, Plasmid Preparation, Sequencing, Binding Assay, Residue

Journal: eLife

Article Title: SIRT2 deacetylase regulates the activity of GSK3 isoforms independent of inhibitory phosphorylation

doi: 10.7554/eLife.32952

Figure Lengend Snippet: ( A ) Western blotting analysis depicting the activity of GSK3 inhibitor X (GSK3-X). Neonatal rat cardiomyocytes were treated with vehicle or 500 nM GSK3-X for 48 hr and the activity of GSK3 was assessed by monitoring the phosphorylation of GS by specific antibody. ( B ) [ 3 H]-leucine incorporation into total cellular protein of control (Ad-GFP) or SIRT2-overexpressing (Ad-SIRT2) rat neonatal cardiomyocytes treated with either vehicle or 500 nM GSK3 inhibitor X (GSK3-X) for 48 hr. Cardiomyocytes were infected with adenoviral vectors encoding either GFP or SIRT2 for 24 hr prior to GSK3-X treatment. After the GSK3-X treatment, cardiomyocytes were stimulated with either vehicle or 20 µM ISO for 24 hr and the [ 3 H]-leucine incorporation was monitored. c.p.m. counts per minute. n = 10. Data is presented as mean ± s.d. *p<0.05. Two-way ANOVA was used to calculate the p values. ( C ) Histogram showing quantification of relative cardiomyocyte area in control (Ad-Null) and SIRT2-overexpressing (Ad-SIRT2) rat neonatal cardiomyocytes treated with either vehicle or 500 nM GSK3 inhibitor X (GSK3-X) for 48 hr. Cardiomyocytes were infected with adenoviral vectors encoding either control or SIRT2 for 24 hr prior to GSK3-X treatment. After the GSK3-X treatment, cardiomyocytes were stimulated with either vehicle or 20 µM ISO for 24 hr and the relative cardiomyocyte area is quantified as described in Materials and methods section. Data is presented as mean ± s.d. *p<0.05. Two-way ANOVA was used to calculate the p values. ( D ) Representative confocal images depicting perinuclear expression of ANP in control (Ad-Null) or SIRT2-overexpressing (Ad-SIRT2) cardiomyocytes treated with either vehicle or ISO (20 µM, 24 hr), with or without GSK3 inhibitor X (GSK3-X, 500 nM, 48 hr). Scale bar = 20 µm. ANP (Green), Myomesin (Red), Hoechst (Blue). ( E ) Western blotting analysis for puromycin incorporation in control or SIRT2-depleted (SIRT2-KD) neonatal rat cardiomyocytes infected with adenovirus expressing either control (Ad-luc shRNA) or p300 shRNA (Ad-p300-shRNA) 48 hr. p300 depletion was confirmed by western blotting. Pulse of puromycin was given 30 min prior to harvesting of cardiomyocytes and puromycin incorporation into nascent proteins was tested using anti-puromycin antibody. # marked Western images denotes SIRT2 antibody used in this assay detects single band. ( F ) Histogram showing relative puromycin levels in control or SIRT2-depleted (SIRT2-KD) cardiomyocytes infected with adenovirus expressing either control (Ad-luc shRNA) or p300 shRNA (Ad-p300-shRNA). The data is generated from . Signal intensities of puromycin and actin were measured by densitometry analysis using ImageJ software. n = 3 independent experiments. Data is presented as mean ± s.d. *p<0.05. Two-way ANOVA was used to calculate the p values. ( G ) Western blotting analysis of GSK3β acetylation and activity in heart lysates of vehicle or anacardic acid (p300 inhibitor) treated 9 months old WT and SIRT2-KO mice littermates. Anacardic acid was injected intraperitoneal at the dose of 5 mg/kg/day for 10 days in mice. Peanut oil was used as vehicle. GSK3β was immunoprecipitated from heart lysates of WT and SIRT2-KO mice using anti-GSK3β antibody (sc-9166, Santa Cruz Biotechnology), and the affinity resin with protein A/G immobilized. Western blotting was performed to detect GSK3β acetylation by anti-Ac-Lysine antibody. GSK3β activity was measured by detecting the phosphorylation of GS. SIRT2 depletion was confirmed by western blotting. Whole cell lysates (WCL) was probed for indicated proteins by western blotting. ( H ) Histogram showing relative GSK3β acetylation in heart lysates of vehicle or anacardic acid (5 mg/kg/day for 10 days) treated 9 months old WT and SIRT2-KO mice from . n = 3. Signal intensities of GSK3β and acetylated-GSK3β was measured by densitometry analysis using ImageJ software. Data is presented as mean ± s.d. *p<0.05. Two-way ANOVA was used to calculate the p values. ( I ) Scatter plot depicting HW/TL ratio of 9 months old WT and SIRT2-KO mice treated with either vehicle or anacardic acid, (p300-INH), at the dose of 5 mg/kg/day for 10 days. n = 5 mice per group. Data is presented as mean ± s.d. *p<0.05. Two-way ANOVA was used to calculate the p values. ( J ) Scatter plot showing left ventricular posterior wall thickness of 9 months old WT and SIRT2-KO mice treated with either vehicle or anacardic acid (p300-INH), at the dose of 5 mg/kg/day for 10 days. n = 6–8 mice per group. Data is presented as mean ± s.d. *p<0.05. Two-way ANOVA was used to calculate the p values. ( K ) Scatter plot depicting cardiac contractile functions, as measured by ejection fraction of 9 months old WT and SIRT2-KO mice treated with either vehicle or anacardic acid (p300-INH), at the dose of 5 mg/kg/day for 10 days. n = 6–8 mice per group. Data is presented as mean ± s.d. *p<0.05. Two-way ANOVA was used to calculate the p values.

Article Snippet: Recombinant protein , p300 , Merck Millipore , 14–418 , http://dx.doi.org/10.1038/s41418-018-0069-8.

Techniques: Western Blot, Activity Assay, Phospho-proteomics, Control, Infection, Expressing, shRNA, Generated, Software, Injection, Immunoprecipitation

Journal: eLife

Article Title: SIRT2 deacetylase regulates the activity of GSK3 isoforms independent of inhibitory phosphorylation

doi: 10.7554/eLife.32952

Figure Lengend Snippet:

Article Snippet: Recombinant protein , p300 , Merck Millipore , 14–418 , http://dx.doi.org/10.1038/s41418-018-0069-8.

Techniques: Knock-Out, Western Blot, Agarose Gel Electrophoresis, Produced, Transfection, Construct, Plasmid Preparation, Modification, Infection, Recombinant, Sequencing, Activity Assay, Mutagenesis, Protease Inhibitor, Microscopy, Software, Membrane, Cell Culture

Journal:

Article Title: Distinct p300-Responsive Mechanisms Promote Caspase-Dependent Apoptosis by Human T-Cell Lymphotropic Virus Type 1 Tax Protein

doi:

Figure Lengend Snippet: (A) Subcellular localizations of wild-type Tax (WT) and truncation mutants in transfected HeLa cells were determined by immunofluorescence microscopy using an anti-Tax rabbit polyclonal antibody. (B) Cytotoxicity induced by Tax mutants was quantified by transient cell death assays. A CMV-Bax expression construct was included as a positive control for apoptosis. Results shown are representative of four independent transfections; error bars represent standard deviations.

Article Snippet: The p300 transcriptional coactivator was detected using a rabbit polyclonal antibody against recombinant human p300 (Santa Cruz Biotechnology, Inc.).

Techniques: Transfection, Immunofluorescence, Microscopy, Expressing, Construct, Positive Control

Journal:

Article Title: Distinct p300-Responsive Mechanisms Promote Caspase-Dependent Apoptosis by Human T-Cell Lymphotropic Virus Type 1 Tax Protein

doi:

Figure Lengend Snippet: (A) HeLa cells were transfected with RcCMV control vector or the CMV–Tax (WT [wild type]), CMV-K88A, CMV-V89A, or CMV-L90A expression construct. Coimmunoprecipitation (IP) was performed using an anti-p300 antibody, and immunocomplexes containing Tax were detected by immunoblotting using an anti-Tax monoclonal antibody. Comparable levels of Tax expression for each mutant were confirmed by Western blot analysis. (B) Tax-derived mutants K88A and V89A exhibited markedly diminished apoptotic potentials in the transient cell death assay compared to the wild-type Tax or L90A mutant. Results represent average percentages derived from three independent experiments. (C) Nuclear fragmentation induced by HTLV-1 Tax is correlated with coactivator binding. HeLa cells were transfected with Tax-expressing vectors and serum starved (0.5% FBS) for 24 h; Tax expression was detected by immunofluorescence microscopy using an anti-Tax monoclonal antibody. Nuclei were visualized by DAPI staining of DNA. Relative average numbers of fragmented nuclei were quantified from three nonoverlapping fields at an original magnification of ×400 from three independent transfections. (D) Immunofluorescence detection of Tax and Tax-derived mutants K88A, V89A, and L90A at an original magnification of ×1,000 (arrows denote nuclear fragmentation bodies). (E) Transactivation of HTLV-LTR-LUC and NF-κB-Luc reporter constructs by wild-type Tax, K88A, V89A, and NLS-ΔN81 proteins in cotransfected HeLa cells; the empty vector is shown as a control. Error bars represent standard deviations.

Article Snippet: The p300 transcriptional coactivator was detected using a rabbit polyclonal antibody against recombinant human p300 (Santa Cruz Biotechnology, Inc.).

Techniques: Transfection, Plasmid Preparation, Expressing, Construct, Western Blot, Mutagenesis, Derivative Assay, Binding Assay, Immunofluorescence, Microscopy, Staining

Journal:

Article Title: Distinct p300-Responsive Mechanisms Promote Caspase-Dependent Apoptosis by Human T-Cell Lymphotropic Virus Type 1 Tax Protein

doi:

Figure Lengend Snippet: (A) Ectopic expression of p300 blocks Tax-induced apoptosis in a dose-dependent manner. HeLa cells were transfected either with an RcCMV control or CMV-Tax expression construct in the presence of increasing concentrations of a CMV-p300 expression vector. β-Galactosidase-expressing cells were detected by staining with an X-Gal solution. (B) Western blot analysis of increased p300-FLAG expression in transfected HeLa cells using a monoclonal antibody against human recombinant p300. (C) Average percentages of Tax-associated cytotoxicity in the presence of increasing concentrations of CMV-p300 were derived from transient cell death assays performed in triplicate. (D) Increased p300 expression did not affect apoptosis induced by C2-ceramide (Sigma) in transient cell death assays; results are representative of duplicate experiments. Error bars indicate standard deviations.

Article Snippet: The p300 transcriptional coactivator was detected using a rabbit polyclonal antibody against recombinant human p300 (Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Transfection, Construct, Plasmid Preparation, Staining, Western Blot, Recombinant, Derivative Assay