Journal: Communications Biology

Article Title: P300-dependent acetylation of the FOXQ1 complex activates super-enhancers to promote colorectal cancer proliferation and metastasis

doi: 10.1038/s42003-025-08430-z

Figure Lengend Snippet: A In HCT-116, Lovo, DLD1 and SW480 cells, FOXQ1 and pan-acetylated-lysine were immunoprecipitated (IP) and examined by western blotting. B Analysis of proteins potentially binding to FOXQ1 using FpClass. C CoIP was used to detect interactions between FOXQ1 and p300. D FOXQ1 was immunoprecipitated and detected for acetylation levels in HEK-293T cells overexpressing p300, GCN5, PCAF and Tip60. E , F HCT-116 and Lovo cells were treated with DMSO or indicated concentration of A485 for 4 h, then acetylation changes of FOXQ1 were detected by immunoprecipitation and western blotting. G Anti-FOXQ1 IPs derived from HCT-116 cells transfected with sip300 or siCTR as negative control, and then the acetylation of FOXQ1 was examined by western blotting. H In vitro acetylation assay using purified Flag-FOXQ1 incubated with p300 in the presence or absence of Acetyl-CoA or A485. I IB analysis of IP products derived from HEK-293T cells transfected with FOXQ1 or KR mutants. J In vitro acetylation dot blotting assay using purified peptides incubated with p300 in the presence or absence of Acetyl-CoA or A485. K In vitro acetylation assay using purified Flag-FOXQ1 incubated with p300 in the presence or absence of Acetyl-CoA to examine the acetylation changes of FOXQ1 Lys190. L Flag-FOXQ1(WT) and Flag-FOXQ1(K190R) were transfected, respectively, into HCT-116 cells. The cytoplasm and nucleus were isolated, and the localization of both in the cells was detected. M DNA pull down was used to detect the effect of K190R on its affinity to the specific binding sequence (TGTTTATGTTTAGTTT) of FOXQ1. N Flag-foxQ1 (WT) and FLAG-K190R (K190R) constructs were transiently transfected into HCT-116 cells, respectively. Subsequently, chromatin immunoprecipitation (ChIP) assays were performed using anti-Flag antibodies to investigate the impact of mutation of FOXQ1-K190 on its interaction with the promoters of SIX2, ZEB1, and STAT2. O The effects of both the acetylated FOXQ1-K190 peptide and the non-acetylated FOXQ1-K190 peptide on their ability to bind to the promoters of SIX2, ZEB1, and STAT2 were investigated using the ChIP assay.

Article Snippet: FOXQ1 (Proteintech, 23718-1-AP, IP: 1:200, WB: 1:1000, IHC: 1:200), p300 (Abcam, ab275378, IP: 1:200, WB: 1:1000), BRD4 (Abcam, ab314432, WB: 1:1000), HDAC1 (Abcam, ab280198, IP: 1:300, WB:1:1000), Flag (Proteintech, 20543-1-AP, WB: 1:10000), Pan Acetylation (Proteintech, 66289-1-Ig, WB:1:1000), RNA-Pol ll (Abcam, ab5408, IP: 1:100, WB:1:1000, ChIP: 1:100), GCN5 (Proteintech, 66575-1-Ig, WB:1:2000), Tip60 (Proteintech, 10827-1-AP, WB:1:2000), PCAF (Proteintech, 28770-1-AP, WB:1:2000), GST (Proteintech, 66001-2-Ig, WB:1:1000), N-Cadherin (Proteintech, 22018-1-AP, WB: 1:2000, IHC: 1:300), Vimentin (Proteintech, 60330-1-Ig, WB: 1:2000, IHC: 1:400), E-Cadherin (Proteintech, 20874-1-AP, WB: 1:2000, IHC: 1:50), Ac-K190 (HUABIO, IHC: 1:200, WB: 1:1000), Ki67 (Proteintech, 27309-1-AP, IHC: 1:200) and Tubulin (Proteintech, 80762-1-RR, WB: 1:20000).

Techniques: Immunoprecipitation, Western Blot, Binding Assay, Concentration Assay, Derivative Assay, Transfection, Negative Control, In Vitro, Acetylation Assay, Purification, Incubation, Isolation, Sequencing, Construct, Chromatin Immunoprecipitation, Mutagenesis

Journal: Cell Death and Differentiation

Article Title: Epigenetic control of mitochondrial cell death through PACS1-mediated regulation of BAX/BAK oligomerization

doi: 10.1038/cdd.2016.119

Figure Lengend Snippet: Transcriptional regulators PCAF or ADA3 control PACS1 expression, and depletion of PACS1 protects against perforin/hGrzB-induced apoptosis.(a) PCAF or ADA3 knockdown (KD) by shRNA significantly downregulates PACS1 expression. Relative PACS1 expression was determined by qPCR in (i) HeLa and (ii) HCT-116 cells that had shRNA-induced KD of PCAF or ADA3 in comparison to shRNA-transduced non-silencing (NS) cells. (b) PCAF and ADA3 are enriched at the PACS1 promoter. Soluble chromatin from HeLa cells was immunopurified with (i) PCAF or (ii) ADA3 antibodies and analysis by qPCR detected enrichment for PCAF or ADA3 at the PACS1 promoter. An IgG control antibody was used for enrichment comparison. (c) PACS1 is depleted in HeLa transduced with shRNA targeting PACS1. HeLa were transduced with shRNA targeted to PACS1 or NS for stable knockdown and (i) relative expression of PACS1 by qPCR was examined and (ii) immunoblot analysis of PACS1 expression was examined. (d) Reduced stable PACS1 expression in HeLa significantly protects cells from perforin/hGrzB-induced cell death compared with NS control. HeLa cells expressing shRNA for NS or PACS1 were treated with sublytic perforin or perforin/hGrzB at the indicated concentrations and analyzed for viability by (i) 51Cr, 4-h release assay or (ii) 24-h survival by AB assay. (e) Transient PACS1-KD in HeLa endows protection from perforin/hGrzB-induced death at a level comparative to stable PACS1-KD. HeLa cells were transfected with siRNA for non-targeting (NT) or PACS1 and (i) 48 h following transfection, relative expression of PACS1 was examined by qPCR. (ii) NT or PACS1-KD cells were treated with sublytic perforin or perforin/hGrzB at the indicated concentrations and a 51Cr, 4-h release assay was performed to examine viability. (f) Perforin/hGrzB-mediated BID cleavage in HeLa with PACS1-KD or NS. HeLa cells were treated with perforin/hGrzB (60 nM) in the absence or presence of the pan-caspase inhibitor QVD (10 μM). Cell lysates for the various conditions shown were analyzed for full length (FL) or cleaved (CL) BID by immunoblot. The immunoblot shown is representative of three independent experiments. (g) Densitometric analysis shows no significant quantitative difference in Bid cleavage in PACS1-KD or NS HeLa cells. The histogram shows the calculated mean ratio of the densitometric signals for CL/FL BID, pooled from three independent experiments (n=3), one of which was the experiment shown in panel (f). Densitometry was quantified using the Image Lab software (Bio-Rad Laboratories). Statistical analyses performed are t-test or one-way ANOVA with Bonferroni test comparing NS to PACS1-KD. NS, non significant, *P<0.05, **P<0.01 and ***P<0.0001

Article Snippet: Immunoprecipitation was performed by adding the soluble chromatin fraction to 10 μ g of either of the following antibodies: rabbit anti-PCAF (A301-666A, Bethyl Laboratories, Montgomery, TX, USA), rabbit anti-ADA3 (ab77011, Abcam, Cambridge, UK), or normal rabbit IgG (ab16640, Abcam), along with protein A-Dynabeads (10001D, ThermoFisher Scientific).

Techniques: Expressing, shRNA, Transduction, Western Blot, Release Assay, Transfection, Software

Journal: The Journal of cell biology

Article Title: Tip60-mediated Rheb acetylation links palmitic acid with mTORC1 activation and insulin resistance.

doi: 10.1083/jcb.202309090

Figure Lengend Snippet: Figure 6. Tip60 interacts with and acetylates Rheb to regulate mTORC1 activity and insulin sensitivity under PA condition. (A) Relative mRNA levels of acetyltransferase genes (cbp, gcn5, pcaf, and tip60) were analyzed by quantitative PCR in fish myocytes under control or 500 μM PA treatment for 12 h (n = 3

Article Snippet: To inhibit CBP/P300, the indicated concentrations of C646 or spermidine were added to the culture medium of C2C12 myotubes for 12 h in the presence of PA. To inhibit GCN5, the indicated concentrations of MB-3 were added to the culture medium of C2C12 myotubes for 12 h in the presence of PA. For MG149 treatment, MG149 (#HY-15887; Med Chem Express) was dissolved in DMSO (Solarbio) to obtain a 150 mM stock solution.

Techniques: Activity Assay, Real-time Polymerase Chain Reaction, Control