Fig 2: Fucoxanthinol decreases the uS7 protein, resulting in G1 cell cycle arrest.HT-29 cells were treated with the indicated concentrations of fucoxanthinol for 6 h, and the protein (a) and mRNA (b) levels of uS7 were examined by western blotting and qRT-PCR analyses. c HT-29 cells were transfected with siCtrl or siuS7 #2. After 48 h, cells were incubated with 10 μM fucoxanthinol for 24 h, and the cell cycle was analyzed by flow cytometry. NT: non-treated, DM: 0.1% DMSO, Data are means ± S.D. (n = 3 biologically independent experiments). *P < 0.05 significantly different from NT.

Fig 3: Ribosomal protein uS7 is the major binding protein of fucoxanthinol.a The scheme for the immobilization of fucoxanthinol onto FG beads® with carboxyl groups. b Whole-cell extracts of HT-29 cells were incubated with empty (−) or fucoxanthinol-fixed (+) FG beads®, and the binding proteins of fucoxanthinol were purified and detected by silver staining. c The major binding protein indicated by an arrowhead in b was analyzed by mass spectrometry, and the molecular masses of the peptides detected are shown. d Whole-cell extracts of HT-29 cells were incubated with fucoxanthinol-fixed beads, and the binding of uS7 was detected by western blotting. e The recombinant His-tagged uS7 protein (His-uS7) was incubated with fucoxanthinol-fixed beads, and the binding of uS7 was detected by western blotting. HOSu: N-hydroxysuccinimide, EDC-HCl: 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, DMAP: 4-dimethylaminopyridine.

Fig 4: uS7 directly binds to CDK6 and is involved in the stability of the CDK6 protein.a HT-29 cells were transfected with siCtrl or siuS7 #2. After 18 h, cells were treated with 20 μg/ml cycloheximide and lysed at the indicated times. The expression levels of CDK2, 4, and 6 were analyzed by western blotting. The sample, designated as “0 h half”, is identical to half the amount of the sample at 0 h. b The expression levels of CDK2, 4, and 6 at each time point were quantified. The expression level at 0 h was defined as 100%. c HT-29 cells were transfected with siCtrl, siuS7 #1, or siuS7 #2. After 24 h, cells were incubated with 10 μM MG-132 for 24 h, lysed, and subjected to western blotting. The signal of each western blot was quantified using ImageJ software and normalized by the value of β-actin. The value of each signal was indicated below the blot. d HT-29 cells were lysed, and the endogenous immunoprecipitation assay was performed with anti-CDK2, CDK4, CDK6, cyclin D, and cyclin E antibodies, respectively. Ribosomal proteins uS7 and uS9 in immunoprecipitates were detected by western blotting. e A GST pull-down assay was performed by incubating the recombinant Myc-DDK-tagged CDK6 protein with GST or GST-fused uS7 (GST-uS7)-bound beads. The CDK6 protein that bound to these beads was detected by western blotting.

Fig 5: The molecular binding model for the interaction between uS7 and fucoxanthinol.a The top-ranked pose in the docking simulation. Fucoxanthinol (green stick) and uS7 (gray cartoon) are shown. b Hydrophobicity around the pocket to which fucoxanthinol binds. Brown and blue colors correspond to the hydrophobic and hydrophilic regions of uS7, respectively. Images (a, b) were made using BIOVIA Discovery Studio 2018 software (Dassault Systèmes, Vélizy-Villacoublay, France).