Fig 2: 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 3: 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 4: Specific inhibition of mTOR pathway by Torin 1 Top: NRCMs treatment with increasing concentration of Torin 1 for 24 h analyzed by immunoblotting for specific signaling pathways. mTORC1 inhibition was assessed by total and phosphorylated levels of rpS6 and 4EBP1. mTOR2 activity was assessed by Akt phosphorylation. Bottom: NRCMs treatment with 100 nM Torin 1 for 3 h analyzed by immunoblotting for specific signaling pathways.Expression of mTOR‐dependent target genes. Expression of eEF2 and rpS5 levels were analyzed by immunoblot (left panel) and RT‐qPCR (right panel) after PE and Torin 1 treatment. Csq—calsequestrin was used as a housekeeping protein. n = 6 biological replicates.mTOR‐dependent expression of hypertrophy markers. RT‐qPCR analysis of Nppa, Nppb, Xirp2 mRNA levels after PE and Torin 1 treatment. Analyzed by one‐way ANOVA. n = 6 biological replicates per conditions.Polysome profiles of control and PE‐ and Torin 1‐treated NRCMs. Data information: Error bars indicate means ± SEM. *P ≤ 0.05 **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Source data are available online for this figure.

Fig 5: 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.