Fig 2: Involvement of CBP and p300 in the transcription of the BRCA1 and RAD51 genes.(A) Down-regulation of BRCA1 and RAD51 proteins upon depletion of CBP and/or p300. H1299 cells were transfected for 48 hr with non-targeting (siNT), CBP (siCBP), p300 (sip300), or CBP+p300 (siC+p) siRNAs. The cells were harvested and whole cell extracts were subjected to immunoblotting. (B, C) Reduction of BRCA1 and RAD51 transcripts in CBP- and p300-depleted cells. H1299 cells were transfected for 48 hr with non-targeting (siNT), CBP (siCBP), p300 (sip300), or CBP+p300 (siC+p) siRNAs. Cells were harvested and subjected to quantitative real-time PCR for the detection of BRCA1 (B) and RAD51 (C) mRNAs. Expression levels were normalized against the levels of GAPDH mRNA. Data represent the mean ± SD. (D) H1299 cells were transfected for 48 hr with non-targeting (siNT), CBP (siCBP), p300 (sip300), or CBP+p300 (siC+p) siRNAs, and stained with propidium iodide (PI). The percentage of cells in each cell cycle phase was determined by FACS. Percentages of cells in G1, S, and G2/M are shown.

Fig 3: The EYA3-p300-SIX5 members bound to the promoters of EGFR, VEGFD, and MMP3. (A) Diagrams of the SIX5 binding site on the promoters (2,000 bp length) of EGFR, VEGFD, MMP8, MMP3, MMP7, MMP21, and MMP26. (B-D) Occupancies of EYA3-p300-SIX5 members on the promoters of EGFR (B), VEGFD (C), and MMP3 (D). Different cells as indicated in the figure were used for ChIP assays with anti-EYA3, anti-p300, anti-SIX5, and IgG (negative control). The input and output DNA were subjected to qRT-PCR analyses to measure the enrichment of EYA3-p300-SIX5 members on the promoters of EGFR, VEGFD, and MMP3. **, P<0.01. ns, no significant difference; KD, knockdown; OE, overexpression; EGFR, epidermal growth factor receptor; VEGFD, vascular endothelial growth factor receptor; MMP, matrix metalloproteinase; ChIP, chromatin immunoprecipitation; qRT-PCR, quantitative real-time polymerase chain reaction.

Fig 4: Schematic representation of the EYA3-SIX5-p300 complex in CRC tumorigenesis and cancer progression. (A) Schematic diagram of the EYA3-SIX5-p300 complex in CRC tumorigenesis and cancer progression. Hypoxia induces EYA3, promoting the assembly of the EYA3-SIX5-p300 complex, which binds to the promoters of EGFR, VEGFD, MMP3, MMP7, MMP8, MMP21, and MMP26. The induction of these target genes triggers CRC tumorigenesis and promotes cancer progression. (B) Schematic diagram of the disruption of EYA3-SIX5-p300 complex by benzarone. Benzarone impairs the assembly of the EYA3-SIX5-p300 complex, causing the suppression of EGFR, VEGFD, MMP3, MMP7, MMP8, MMP21, and MMP26, and attenuating tumor growth. CRC, colorectal cancer; EGFR, epidermal growth factor receptor; VEGFD, vascular endothelial growth factor receptor; MMP, matrix metalloproteinase.

Fig 5: Activation of melatonin receptor signaling preserves p300 levels in beta-cells exposed to diabetes-related stress.a INS-1E cells were exposed to 1, 10, 100 nM melatonin during 24 h (C, control cells). Protein levels of p300 and acetylated histone H4 (Ac-H4) were analyzed by western blot. GAPDH was used as loading control. The graphs represent the quantification of p300 and Ac-H4 protein levels (n = 3–4). b p300 and actin (loading control) protein levels in INS-1E cells cultured in 11 mM glucose (G11) or 30 mM glucose (G30) and exposed for the final 14 h to media containing melatonin (100 nM) or not. The graphs represent the quantification of p300 protein levels (n = 3). c p300 and actin (loading control) protein levels in INS-1E cells exposed or not to pro-inflammatory cytokines mix (CK: 50 ng/ml TNFα, 0.2 ng/ml IL1β and 33 ng/ml IFNγ for 24 h (C, control) and incubated for the final 14 h to media containing melatonin (100 nM) or not. The graphs represent the quantification of p300 protein levels (n = 3). Data are expressed as mean ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001. NS, non-significant