Fig. 4: Phosphorylation of S1042 determines the cytoplasmic distribution of TRIM24.

A Immunohistochemical staining of TRIM24 in CRC tissues (n = 12) obtained from the Human Protein Atlas database (https://www.proteinatlas.org/). Shown are representative images of strict nuclear localization (N: 5/12) and partially cytoplasmic distribution (C + N: 7/12) of TRIM24, respectively. B Schematic diagram of human TRIM24, predicted nuclear localization sequences (NLS1 and NLS2) using cNLS Mapper, the mutant amino acid sites of NLSs (NLS1-M and NLS2-M), and the mutants of TRIM24 at serine 1042 (S1042). C The structure of human TRIM24 protein (left) and its carboxyl terminus (right) were obtained from AlphaFold3 (UniProt ID: O15164). The S1042 residue is illustrated in orange (right). D The amino acid sequences of TRIM24 from human, mouse, rat, and monkey were aligned, with the potential phosphorylation site S1042 (or S1043) highlighted in bold. E Immunofluorescence micrographs of HEK293T cells transfected with GFP-TRIM24 plasmid or TRIM24 mutant plasmid as shown in (B), with the nucleus counterstrained with DAPI. Scale bar, 10 µm. F The statistical analysis based on TRIM24 distribution shows localization in the nucleus (N) or in both the cytoplasm and nucleus (C + N) within HEK293T cells in (E). The subcellular localization of GFP and GFP-fusion proteins was quantified separately in five randomly selected fields for each group. Experiments were conducted independently three times, consistently producing similar results. G Normal human colonic epithelial cell line FHC and human CRC cell lines were harvested and immunoblotted for indicated proteins, with Actin as a loading control. Data are representative of at least three independent experiments. H The cytosolic and nuclear fractions of CRC cell lines were extracted and immunoblotted for indicated proteins. GAPDH and Lamin-A were employed as loading controls of cytosol fraction and nucleus fraction, respectively, and indicated the purity of each fraction. Data are representative of at least three independent experiments. I Left: HT29 cell were transfected with GFP, GFP-TRIM24, or TRIM24 mutant plasmids. After 36 h, whole cell lysates were derived and immunoblotted for indicated proteins. Right: The levels of indicated proteins, normalized to vehicle controls, were quantified from three independent experiments. Data were expressed as means ± SEM, two-tailed Student’s t-test. J TRIM24 S1042 phosphorylation in DLD1 cells was detected by label-free relative quantitative mass spectrum analysis.