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