Fig 1: AA and U0126 alleviates PE-induced cardiomyocyte hypertrophy. (A-C) Western blotting data for cardiac hypertrophy biomarkers (ANP, BNP and ß-MHC). (D) Cell surface area was measured by immunofluorescence staining to demonstrate the hypertrophic responses in cardiomyocytes. (E) Quantification of the cell surface area of myocardial cells. Scale bar, 20 µm. n=6. *P<0.05 vs. control group; #P<0.05 vs. PE group. PCAF, P300/CBP-associated factor; PE, phenylephrine; AA, anacardic acid; PE, 100 µmol/l phenylephrine for 48 h; Vehicle, 100 µmol/l phenylephrine + equal volume of DMSO for 48 h; AA, 50 µmol/l AA for 30 min + 100 µmol/l phenylephrine for 48 h; AA + U, 50 µmol/l AA + 10 µmol/l U0126 for 30 min + 100 µmol/l phenylephrine for 48 h; U, 10 µmol/l U0126 for 30 min + 100 µmol/l phenylephrine for 48 h; Control, no drug; ANP, atrial natriuretic peptide; ß-MHC, ß-myosin heavy chain; BNP, brain natriuretic peptide; PE, phenylephrine.
Fig 2: Effect of KLF4 acetylation by PCAF on IL-6 gene transcription in GMCs exposed to sublytic C5b-9. GMCs were transfected with wild-type PCAF (PCAF WT), PCAF HAT-deficient mutant (PCAF?HAT), wild-type KLF4 (KLF4 WT), acetylation-deficient mutant (KLF4-K224R), or hyper-acetylated mutant (KLF4-K224Q) for 48 h. (A) ELISA of IL-6 in supernatants of GMCs. (B) qPCR analysis of IL-6. (C) Luciferase activity assay of IL-6 reporter (-1,791 to +30 nt). (D) ChIP–qPCR analysis of KLF4 enrichment on IL-6 promoter (-372 to -166 nt). **p < 0.01 vs. PCAF WT + KLF4 WT. Data are shown as mean ± SD from three independent experiments. GMCs, glomerular mesangial cells; ChIP, chromatin immunoprecipitation.
Fig 3: Effect of histone acetylation by PCAF on IL-6 gene transcription in GMCs in response to sublytic C5b-9. (A–D) GMCs were transfected with shCTR or shPCAF for 48 h and then incubated with sublytic C5b-9 for 3 h. (A) IB analysis of H2B acetyl K5, H2B acetyl K15, H2B acetyl K20, and H2B. (B) IB analysis of H3 acetyl K9, H3 acetyl K14, H3 acetyl K18, and H3. (C) ChIP–qPCR analysis of enrichment of H2B acetyl K15 on IL-6 promoter (-372 to -166 nt). (D) ChIP–qPCR analysis of enrichment of H3 acetyl K9 on IL-6 promoter (-372 to -166 nt). **p < 0.01 vs. shCTR + sublytic C5b-9. (E–G) GMCs were pretreated with DMSO or anacardic acid (30 µM) for 1 h and then stimulated with sublytic C5b-9 for 3 h. (E) IB analysis of IL-6, H2B acetyl K5, and H3 acetyl K9. (F) qPCR analysis of IL-6. (G) Luciferase activity assay of IL-6 reporter (-1,791 to +30 nt). **p < 0.01 vs. DMSO + sublytic C5b-9. (H) GMCs were pretreated with BAY 11-7082 for 30 min and then stimulated with sublytic C5b-9 for 3 h. IB analysis of KLF4 (**p < 0.01 vs. DMSO + sublytic C5b-9). (I) A putative scheme for regulatory mechanisms of IL-6 expression induced by sublytic C5b-9 in GMCs. KLF4 binds to IL-6 promoter and activates its transcription. PCAF acetylates KLF4 (at K224) and neighboring histones H2B (at K5) and H3 (at K9), contributing to IL-6 transcription. Besides, NF-?B signaling pathway also regulates IL-6 expression via increasing PCAF expression. Data are representative of three independent experiments with similar results or are shown as mean ± SD from three independent experiments. GMCs, glomerular mesangial cells; IB, immunoblotting; DMSO, dimethyl sulfoxide.
Fig 4: p-ERK1/2 interacted with PCAF and modified H3K9ac acetylation in hypertrophic cardiomyocytes induced by PE. (A) Different concentrations of AA (30, 40, 50 and 60 µmol/l) were used to identify the optimal concentration of AA; 50 µmol/l was selected for subsequent experiments, based on the levels of histone H3K9ac. (B) Effects of different concentrations of the ERK inhibitor U0126 (2, 4, 6, 8 and 10 µmol/l) on cell viability in neonatal mouse cardiomyocytes. (C) Expression of T-ERK1/2 and p-ERK1/2 in myocardial cells from neonatal mice. (D) Co-immunoprecipitation in cell lysates of mouse myocardial cells exposed to six different experimental conditions with anti-p-ERK1/2-protein G magnetic beads and IB with anti-PCAF, anti-H3K9ac or anti-p-ERK1/2 antibodies to evaluate protein expression. Input, positive control; IgG, negative control. n=6. *P<0.05 vs. control group; #P<0.05 vs. PE group. P-, phospho-; PCAF, P300/CBP-associated factor; PE, phenylephrine; H3K9ac, histone 3 acetylation K9; AA, anacardic acid; T-, total-; IB, immunoblotting; ERK, extracellular signal-regulated protein kinase; PE, 100 µmol/l phenylephrine for 48 h; Vehicle, 100 µmol/l phenylephrine + equal volume of DMSO for 48 h; AA, 50 µmol/l AA for 30 min + 100 µmol/l phenylephrine for 48 h; AA + U, 50 µmol/l AA + 10 µmol/l U0126 for 30 min + 100 µmol/l phenylephrine for 48 h; U, 10 µmol/l U0126 for 30 min + 100 µmol/l phenylephrine for 48 h; Control, no drug.
Fig 5: PCAF binding, acetylation levels of H3K9ac in the MEF2C promoter and the expression of MEF2C in myocardial cells. (A) Binding of PCAF to the promoter in MEF2C was assessed using ChIP-qPCR. (B) Acetylation levels of histone H3K9ac in the promoter of MEF2C were assessed using ChIP-qPCR. (C) mRNA expression of MEF2C in cardiomyocytes exposed to six different conditions. (D) ChIP-qPCR data showed that the cardiac nuclear transcription factor MEF2C could bind to the promoter of biomarker genes for cardiac hypertrophy (ANP, BNP and ß-MHC). n=6. *P<0.05 vs. control group; #P<0.05 vs. PE group. PCAF, P300/CBP-associated factor; PE, phenylephrine; AA, anacardic acid; PE, 100 µmol/l phenylephrine for 48 h; Vehicle, 100 µmol/l phenylephrine + equal volume of DMSO for 48 h; AA, 50 µmol/l AA for 30 min + 100 µmol/l phenylephrine for 48 h; AA + U, 50 µmol/l AA + 10 µmol/l U0126 for 30 min + 100 µmol/l phenylephrine for 48 h; U, 10 µmol/l U0126 for 30 min + 100 µmol/l phenylephrine for 48 h; Control, no drug; PCAF, P300/CBP-associated factor; PE, phenylephrine; H3K9ac, histone 3 acetylation K9; MEF2C, myocyte enhancer factor 2C; ChIP-qPCR, chromatin-immunoprecipitation-quantitative PCR; ANP, atrial natriuretic peptide; ß-MHC, ß-myosin heavy chain; BNP, brain natriuretic peptide.
Supplier Page from Abcam for Anti-KAT2B / PCAF antibody [EPR2670(N)]