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: 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: 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

Journal: Advanced Science

Article Title: Chemotherapy‐Mediated Induction of PD‐L1 via SEI1 Facilitates Myeloma Immune Evasion

doi: 10.1002/advs.202411082

Figure Lengend Snippet: SEI1 bridges the interaction between CBP/p300 and PAF1 complex to regulate PD‐L1 transcription. A) HA‐SEI1 overexpressed IM‐9 cells were lysed, and the total protein lysate was immunoprecipitated with an agarose‐immobilized HA antibody and analysis with mass spectrometry. The proteins identified are indicated. B,C) Co‐immunoprecipitation of SEI1, PAF1, CDC73, or p300 in ARH‐77 or IM‐9 cells treated with melphalan or bortezomib. D) Co‐immunoprecipitation of SEI1 along with PAF1, CDC73, or p300 in HEK293T cells co‐transfected with SEI1 and either PAF1 , CDC73 , or p300 plasmid. E) Schematic of the truncations including ΔCABD, ΔSD, ΔPBID, and ΔCT fragments. F) Western blotting showing different truncations of SEI1 (full length, ΔCABD, ΔSD, ΔPBID, and ΔCT) in HEK293T cells. G–K) Pulldown of PAF1, CDC73, or p300 with different truncations of SEI1 (full length, ΔCABD, ΔSD, ΔPBID, and ΔCT) in HEK293T cells. L) Pulldown of MYC‐p300 with CDC73 or PAF1 in HEK293T cells. M) Gene tracks showing representative ATAC‐seq profiles at PD‐L1 gene loci in primary CD138 + plasma cells from two relapsed myeloma patients following treatment with bortezomib. N,O) ChIP PCR assay showing SEI1, p300, or H3K27ac enrichment on PD‐L1 promoter of ARH‐77 or IM‐9 cells treated with or without melphalan or bortezomib. P) ChIP‐seq profiles show H3K27ac signal tracks at the PD‐L1 gene loci for ARH‐77 cells treated with or without melphalan or bortezomib. Q) ChIP PCR assay showing H3K27ac enrichment on PD‐L1 promoter of ARH‐77 or IM‐9 cells treated with or without melphalan or bortezomib. R) ChIP PCR assay showing enrichment of H3K27ac on PD‐L1 promoter in sh Ctrl or sh SEI1 ARH‐77 or IM‐9 cells. S) Expression of PAF1 in nonspecific, PAF1 shRNA‐expressing myeloma cells. T,U) The relative expression of PD‐L1 in PAF1 knockdown ARH‐77 and IM‐9 cells treated with or without melphalan or bortezomib. V,W) ChIP PCR assay showing enrichment of V) SEI1 or W) H3K27ac on PD‐L1 promoter in sh Ctrl or sh PAF1 ARH‐77 and IM‐9 cells. X) Schematic of the SEI1 bridges the interaction between CBP/p300 and PAF1 complex to regulate PD‐L1 transcription. B–D,F–L,N,O,Q–W) Representative of three independent experiments. Data are averages ± SD. *** p < 0.001 and **** p < 0.0001. All p ‐values were determined using one way ANOVA.

Article Snippet: Chromatin samples were immunoprecipitated with antibodies against phosphorylated IRF7 (#12390), acetyl‐histone H3 (Lys27) (H3k27ac) (#8173), p300 (#54062), phosphorylated p65 (#3033), trimethyl‐histone H3 (Lys4) (H3K4me3) (#9751), trimethyl‐histone H3 (Lys27) (H3k27me3) (#9733), trimethyl‐histone H3 (Lys36) (H3k36me3) (#4909), phosphorylated IRF3 (#29 047) (Cell Signaling Technology), SEI1 (sc‐517080, Santa Cruz), and control IgG at 4 °C for 3 h. Immunoprecipitates and total chromatin inputs were reverse cross‐linked; DNA was isolated and analyzed using PCR with primers.

Techniques: Immunoprecipitation, Mass Spectrometry, Transfection, Plasmid Preparation, Western Blot, Clinical Proteomics, ChIP-sequencing, Expressing, shRNA, Knockdown

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