Journal: Cell Death & Disease

Article Title: FK506 binding protein 51 positively regulates melanoma stemness and metastatic potential

doi: 10.1038/cddis.2013.109

Figure Lengend Snippet: FKBP51 interacts with the general transcriptional co-activator p300. ( a , left) FKBP51 co-immunoprecipitates with p300. ( a , right), p300 co-immunoprecipitates with FKBP51. Total-cell lysates were prepared by SAN melanoma cells transfected with FKBP51/Flag. Cell lysates were immunoprecipitated with anti-Kat3B/p300 (IP p300) or anti-Flag (IP FKBP51). Immunoprecipitated and total lysates were then subjected to western blot with anti-FKBP51 or anti-p300. ( b ) ChIP performed with SAN melanoma cells, silenced (FKBP51 siRNA) or not (NS RNA) for FKBP51. An enrichment of DNA (region at −3450 from TSS of ABCG2 gene) can be observed in p300-immunoprecipitated chromatin (NS RNA) compared with IgG sample. Such an enrichment appeared to be reduced when FKBP51 was silenced (FKBP51 siRNA)

Article Snippet: The p300- immunoprecipitated chromatin was obtained using KAT3B/p300 Antibody (RW109) (Novus Biologicals).

Techniques: Transfection, Immunoprecipitation, Western Blot

Journal: RNA

Article Title: Regulation of alternative splicing by p300-mediated acetylation of splicing factors

doi: 10.1261/rna.069856.118

Figure Lengend Snippet: p300 is a regulator of both transcription and alternative splicing. MCF7 cells were transfected with nontargeting siRNA (siNT) and sip300 for 72 h. RNA was extracted, libraries prepared, and RNA-seq was conducted. ( A ) Venn diagram representing differentially expressed genes (light green) and alternatively spliced genes (light orange). ( B , C ) Functional analysis was conducted using the DAVID functional annotation tool (DAVID, https://david.ncifcrf.gov/ ) for ( B ) differentially expressed genes and ( C ) alternative splicing genes. ( D ) Real-time PCR analysis of total mRNA amount of CD44 relative to CycloA reference gene. ( E ) Real-time PCR analysis of CD44s and CD44v5-6 relative to CD44 total mRNA amount. PSI was calculated by CD44v5-6/CD44s + CD44v5-6. Values represent averages of three independent experiments ± SE; (**) P < 0.01 (paired Student's t -test).

Article Snippet: After 30 h in culture, plasmid transfected cells were used for experimentation. pcDNA-dCas9-p300 Core plasmids (D1399Y; plasmid #61358 and plasmid #61357) were purchased from Addgene. pSPgRNA (Addgene, plasmid #47108) was used as the gRNA plasmid.

Techniques: Alternative Splicing, Transfection, RNA Sequencing, Functional Assay, Real-time Polymerase Chain Reaction

Journal: RNA

Article Title: Regulation of alternative splicing by p300-mediated acetylation of splicing factors

doi: 10.1261/rna.069856.118

Figure Lengend Snippet: Tethering p300 to CD44 promoter up-regulates its expression and promotes CD44v inclusion. ( A – C ) HEK293T cells were transfected with either dCas9-p300 core (mut) or dCas9-p300 core (WT) with four gRNAs targeted to the CD44 promoter, Intron 8 (In8) or 3′ UTR for 30 h. Total RNA was extracted and analyzed by real-time PCR for total mRNA amount of CD44 relative to CycloA reference gene ( A ) and for total mRNA amount of CD44s and CD44v6-7 relative to CD44 total mRNA amount ( B ). PSI was calculated by CD44v6-7/CD44s + CD44v6-7. ChIP was performed of H3 pan-acetylated, along the CD44 gene ( C ). Values represent averages of three independent experiments ± SE and are expressed as dCas9-p300 core (WT) relative to dCas9-p300 core (mut) (*) P < 0.05, (**) P < 0.01, for gRNA to promoter; (#) P < 0.05 gRNA to In8; (†††) P < 0.005 gRNA to 3′ UTR; Student's paired t -test. ( D , E ) HEK293T cells were transfected with either WT RNAPII or Slow RNAPII and after 24 h treated with α-amanitin for 24 h ( D ); or cells were treated with 6 µM CPT for 6 h ( E ). Total RNA was extracted and analyzed by real-time PCR for CD44s and CD44v6-7 relative to CD44 total mRNA amount. PSI was calculated by CD44v6-7/CD44s + CD44v6-7. Values represent mean ± SE of seven ( D ) or three ( E ) independent experiments.

Article Snippet: After 30 h in culture, plasmid transfected cells were used for experimentation. pcDNA-dCas9-p300 Core plasmids (D1399Y; plasmid #61358 and plasmid #61357) were purchased from Addgene. pSPgRNA (Addgene, plasmid #47108) was used as the gRNA plasmid.

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

Journal: RNA

Article Title: Regulation of alternative splicing by p300-mediated acetylation of splicing factors

doi: 10.1261/rna.069856.118

Figure Lengend Snippet: p300 acetylates Sam68 and hnRNP M. ( A ) Enrichment of binding sites of six of CD44's known splicing factors in p300 regulated exons: SAM68, hnRNP M, hnRNP L, TRA2β, hnRNP F, hnRNP A1. Shown are the −lg ( P -values) enrichment of binding sites in skipped exons versus included ( right bars) and included versus skipped ( left bars). Dashed red line denotes significant P -value (corrected for multiple testing, Bonferroni correction). As can be seen, the binding sites of SAM68 and hnRNP M were found to be significantly enriched in the skipped exons. ( B , C ) HEK293T cells were transfected with nontargeting siRNA (siNT) and sip300 for 72 h. Sam68 was immunoprecipitated and acetylated lysine (AK) was detected ( B ). (*) Heavy chain of Sam68 Ab. hnRNP M was immunoprecipitated and acetylated lysine (KA) was detected ( C ). ( D ) MCF7 cells were transfected with nontargeting siRNA (siNT) or siRNA against p300 (sip300) or against Sam68 (siSam68) or hnRNP M (sihnRNP M) for 72 h. Total RNA was extracted and analyzed by real-time PCR for CD44s and CD44v5-6 relative to CD44 total mRNA amount. PSI was calculated by CD44v5-6/CD44s + CD44v5-6. Values represent averages of three independent experiments ± SE. (*) P < 0.05; (***) P < 0.001; Student's paired t -test. ( E ) HEK293T transfected with nontargeting siRNA (siNT) or siRNA against p300 (sip300) and transfected again after 48 h with either pcDNA3-empty vector or hnRNP M for 24 h. Total RNA was extracted and analyzed by real-time PCR for total mRNA amount of CD44s and CD44v6-7 relative to CD44 total mRNA amount. PSI was calculated by CD44v6-7/CD44s + CD44v6-7. Values represent averages of three independent experiments ± SE.

Article Snippet: After 30 h in culture, plasmid transfected cells were used for experimentation. pcDNA-dCas9-p300 Core plasmids (D1399Y; plasmid #61358 and plasmid #61357) were purchased from Addgene. pSPgRNA (Addgene, plasmid #47108) was used as the gRNA plasmid.

Techniques: Binding Assay, Transfection, Immunoprecipitation, Real-time Polymerase Chain Reaction, Plasmid Preparation

Journal: RNA

Article Title: Regulation of alternative splicing by p300-mediated acetylation of splicing factors

doi: 10.1261/rna.069856.118

Figure Lengend Snippet: p300 excludes hnRNP M from CD44 pre-mRNA and promotes cell motility. ( A ) RNA-IP of hnRNP M in HEK293T cells transfected with either dCas9-p300 core (mut) or dCas9-p300 core (WT) with four gRNAs targeted to the CD44 promoter. Real-time PCR analysis of CD44 intron 8. Values represent averages of three independent experiments ± SE. ( B ) hnRNP M was immunoprecipitated from HEK293T cells and interacting proteins were detected with indicated antibodies. ( C ) HDAC1 was immunoprecipitated from HEK293T cells and interacting proteins were detected with indicated antibodies. (*) Long exposure. ( D ) HEK293T cells were transfected with either dCas9-p300 core (mut) or dCas9-p300 core (WT) with four gRNAs targeted to the CD44 promoter for 30 h. ChIP was performed on HDAC1, along the CD44 gene. ( E ) HEK293T cells were treated with 10 µM TSA for 2 h. Real-time PCR analysis of CD44s and CD44v5-6 relative to CD44 total mRNA amount. PSI was calculated by CD44v5-6/CD44s + CD44v5-6. ( F , G ) MCF7 cells stable for dCas9-p300 core (mut) or dCas9-p300 core (WT) were transfected with four gRNAs targeted to CD44 promoter or 3′ UTR for 30 h. Cells were seeded in 96-well plates and a scratch assay was conducted. Results were calculated for gRNA to promoter relative to 3′ UTR. Values represent averages of three independent experiments ± SE. ( G ) Western blot was conducted using the indicated antibodies.

Article Snippet: After 30 h in culture, plasmid transfected cells were used for experimentation. pcDNA-dCas9-p300 Core plasmids (D1399Y; plasmid #61358 and plasmid #61357) were purchased from Addgene. pSPgRNA (Addgene, plasmid #47108) was used as the gRNA plasmid.

Techniques: Transfection, Real-time Polymerase Chain Reaction, Immunoprecipitation, Wound Healing Assay, Western Blot

Journal: RNA

Article Title: Regulation of alternative splicing by p300-mediated acetylation of splicing factors

doi: 10.1261/rna.069856.118

Figure Lengend Snippet: Schematic representation of the proposed mechanism. The chromatin factor p300 acetylates histones as well as splicing factors to regulate, respectively, transcription and alternative splicing.

Article Snippet: After 30 h in culture, plasmid transfected cells were used for experimentation. pcDNA-dCas9-p300 Core plasmids (D1399Y; plasmid #61358 and plasmid #61357) were purchased from Addgene. pSPgRNA (Addgene, plasmid #47108) was used as the gRNA plasmid.

Techniques: Alternative Splicing

Journal: Cell proliferation

Article Title: Elevated COMMD1 Contributes to Cardiomyocyte Copper Efflux in Chronic Myocardial Ischemia: Insights From Rhesus Monkey.

doi: 10.1111/cpr.70016

Figure Lengend Snippet: FIGURE 3 | Increased XIAP associated with COMMD1 in cardiomyocytes after chronic LAD ligation in rhesus monkeys. (A) The mRNA levels of COMMD1 determined by RT-PCR. (B) Western blot and (C) quantitative analysis of the protein levels of p300 and XIAP in different portions of the heart of rhesus monkey (Sham: n = 4; MI: n = 4). (D) Immunofluorescence image and (E) co-localization analysis showing COMMD1 (green) or XIAP (green) with cardiomyocytes (α-actinin, red) in the serial sections of the heart of rhesus monkey, DAPI stain (blue) labels nuclei, scale bar = 100 μm. (F) Immunoprecipitation-Western blotting analysis of COMMD1 and XIAP in the ischemic heart tissue of rhesus monkeys and the control res- in + mouse antibody against COMMD1 or coupling resin + mouse IgG as the control groups. (G) Western blotting analysis of the protein levels of COMMD1 and XIAP in the cardiac tissues of rhesus monkeys after 2 and 24 h of LAD ligation (Sham: n = 4; 2 h MI: n = 2; 24 h MI: n = 2). (H) The ratio of XIAP versus COMMD1 protein levels in the heart of rhesus monkeys. Data were expressed as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 versus Sham control. IA, infarct area; RA, remote area.

Article Snippet: PVDF membranes were incubated overnight at 4°C with the respective primary antibodies: monoclonal mouse anti- CCS (1:100, SC- 55560, Santa cruz, USA), monoclonal mouse anti- COMMD1 (1:100, SC- 166248, Santa cruz, USA), polyclonal rabbit anti- COMMD1 (1:1000, A5174, Abclonal, China), monoclonal mouse anti- XIAP (1:100, SC- 55551, Santa Cruz, USA), polyclonal rabbit anti- XIAP (1:1000, NB- 100- 56,183, Novus, USA), monoclonal rabbit antiATP7A (1:1000, ab308524, abcam, USA), monoclonal rabbit anti- ATP7B (1:1000, ab131208, abcam, USA), monoclonal mouse anti- GAPDH (1:1000, TA- 08, Zhongshan, China) and monoclonal rabbit anti- p300 (1:1000, 86,377, CST, USA).

Techniques: Ligation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Immunofluorescence, Staining, Immunoprecipitation, Control

Journal: The Journal of experimental medicine

Article Title: PARP7 inhibits type I interferon signaling to prevent autoimmunity and lung disease.

doi: 10.1084/jem.20241184

Figure Lengend Snippet: Figure 7. PARP7 inhibits the interaction between IRF3 and transcriptional co-activators CBP/p300. (A) Co-immunoprecipitation analysis of endogenous IRF3:CBP/p300 interaction. WT and Parp7−/−MEFs were stimulated with DMXAA (10 μg/ml) for the indicated time points, followed by IP with IRF3 antibody and blotted with the indicated antibodies. (B) Co-immunoprecipitation analysis of IRF3:CBP/p300 interaction. HEK293T cells were transfected with HA-IRF3- 5D and EGFP-PARP7 or EGFP-PARP7-H532A, followed by IP with HA antibody and blotted with indicated antibodies. (C) A heat map showing the expression of various ISGs following treatment with increasing doses of A-485 (0, 1, 5, and 10 μM, 24 h), DMXAA (10 μg/ml, 4 h), or RBN-2397 (1 μM, 24 h). (D) Representative bar graphs of individual ISGs from D. Data are shown as mean ± SD. P values were determined by two-way ANOVA (E). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns, not significant. Data are representative of three independent experiments. Source data are available for this figure: SourceData F7.

Article Snippet: Primary antibodies used for western blot included phospho-TBK1 (5483; CST), TBK1 (3504; CST), phospho-IRF3 (4947; CST), IRF3 (4302; CST), phospho-STING (72971; CST), STING (13647; CST), phospho-STAT1 (9167; CST), STAT1 (9172; CST), IFN-β (CST; 97450), p300 (57625; CST), CBP (7389; CST), GAPDH (2118; CST), alpha-Tubulin (T5168; Sigma-Aldrich), β-Actin (4967; CST), Lamin A/C (2032; CST), Histone H3 (4499; CST), FLAG (14793; CST), HA (3724; CST), GFP (A11122; Invitrogen), andmono/polyADPr (83732; CST).

Techniques: Immunoprecipitation, Transfection, Expressing

Journal: Biomedicines

Article Title: R -α-Lipoic Acid and 4-Phenylbutyric Acid Have Distinct Hypolipidemic Mechanisms in Hepatic Cells

doi: 10.3390/biomedicines8080289

Figure Lengend Snippet: PBA enhances the acetylation of CBP/p300 and histone H3 at the CPT1A and INSIG2 promoters in Huh7-shTSC2 cells. PBA (8 mM, 6 h) induced the gene expression of CPT1A and INSIG2 ( A ), and the acetylation status of histone H3 and histone H4 ( B ). Chromatin immunoprecipitation (ChIP) assay revealed PBA-mediated enrichment of acetyl CBP/p300 (Ac-p300) and acetyl histone H3 (Ac-H3) at the CPT1A and INSIG2 promoters ( C ). Statistical significance was determined using Student’s t -test, * p < 0.05, n = 4.

Article Snippet: After pre-clearing the sheared chromatin, samples were subjected to immunoprecipitation using the following the antibodies: anti-acetyl-CBP [Lys1535]/p300 [Lys1499] (Cell Signaling Technology #4771S, Danvers, MA, USA), anti-acetyl-histone H3 (Millipore #06–599, Burlington, MA, USA), anti-histone H3 (Millipore #07–690).

Techniques: Gene Expression, Chromatin Immunoprecipitation