Fig 1: Urolithin A promotes ERK1/2 activation by directly binding to ERK1/2. A) UA‐conjugated Fe3O4 beads (UA‐Beads) were used to pull down target protein from Jurkat cells in vitro, followed by proteomic mass spectrometry analysis. Mass spectrometry results are from one experiment with three technical replicates per sample. B) Immunoblot analysis of ERK1/2 and MEK1 pulled down by UA‐beads from CD8+ CTL lysate. The pull‐down assay was repeated three times independently. C) In vitro binding assay of UA‐beads with ERK1, ERK2, and MEK1. Commercial purified Flag‐ERK1, Flag‐ERK2, and MEK1 proteins were incubated overnight with Fe3O4 control beads or UA‐beads at 4 °C. The proteins were then boiled with an SDS‐loading buffer and subjected to Western blot analysis. This experiment was repeated three times independently. D) SPR assay for the affinity between UA and purified his tagged ERK1 protein. SPR assay data are from one experiment with three technical replicates per sample. E) CTESA assay with UA‐treated CTLs. CD8+ CTLs were exposed to 10 µm UA (DMSO as control) for 24 h and heated at a specified temperature gradient (42° to 64 °C) for 3 min to denature the proteins, followed by Western blot analysis of ERK1/2. This experiment was repeated three times independently. F) CD8+ CTLs were treated with DMSO and UA (10 µm) for 48 h and stimulated with anti‐CD3/CD28 antibodies for 10 min, followed by immunoblot analysis of indicated proteins. Representative immunoblot image (left) and quantification of the indicated protein (right, normalized to total protein) are shown. Data are presented as means ± SEM (n = 3) and were analyzed by two‐tailed unpaired Student's t‐test. This experiment was repeated three times independently. G) Inactive ERK1/2 and activated MEK were incubated with UA (10 µm) and ATP for in vitro kinase assay. The reactions were subjected to immunoblot analysis with indicated antibodies. This experiment was repeated three times independently. Quantification analysis data are presented as means ± SEM (n = 3) and were analyzed by two‐tailed unpaired Student's t‐test. H) HEK293T cells were transfected with plasmids expressing the indicated HA‐tagged deletion mutant of ERK1 and then used to perform a pull‐down assay with control beads or UA‐beads. This experiment was repeated three times independently. I) Tat‐tagged ERK1156‐166 (Tat‐ERK) peptide (+, 5 µm; ++, 10 µm) was synthesized and used to compete for the pull‐down of commercial FLAG‐ERK1 with UA‐Beads. This experiment was repeated three times independently. Quantification analysis data are presented as means ± SEM (n = 3) and were analyzed by two‐tailed unpaired Student's t‐test. J) OT‐I CD8+ CTLs were treated with DMSO or UA (10 µm) in the presence or absence of Tat‐ERK (10 µm) for 48 h and stimulated with anti‐CD3/CD28 antibodies for 10 min, followed by immunoblot analysis of p‐ERK1/2 and ERK1/2. This experiment was repeated three times independently. Quantification of p‐ERK1(right, normalized to total ERK1/2) are presented as means ± SEM (n = 3) and were analyzed by two‐tailed unpaired Student's t‐test. K) OT‐I CTLs were treated with DMSO or UA (10 µm) in the presence or absence of Tat‐ERK (10 µm) for 48 h and then stimulated with anti‐CD3/CD28 antibodies for 6 h. Production of IFN‐γ, TNF‐α, IL‐2, and granzyme B in CD8+ CTLs was assessed using flow cytometric analysis. Data are presented as means ± SEM (n = 3) and were analyzed by two‐tailed unpaired Student's t‐test. This experiment was repeated three times independently. L) OT‐I CTLs were treated with DMSO or UA (10 µm) in the presence or absence of Tat‐ERK (10 µm) for 48 h. Cytotoxicity of the treated CTLs against 10 nm OVA257‐264 peptide‐pulsed EL4 targets was determined in vitro. Data are presented as means ± SEM (n = 3) and were analyzed by two‐tailed unpaired Student's t‐test. M) OT‐I CTLs were treated with DMSO or UA (10 µm) in the presence or absence of Tat‐ERK (10 µm) for 48 h. The percentage of CD62L+CD44+ CD8+ T cells was evaluated using flow cytometric analysis. Data are presented as means ± SEM (n = 3) and were analyzed by two‐tailed unpaired Student's t‐test. N–Q) OT‐I CTLs were treated with DMSO or UA (10 µm) in the presence of Tat‐ERK (10 µm) for 48 h and transferred into B16‐MO5‐Fluc lung metastases‐bearing C57BL/6 mice. Schematic diagram of the experiment (N). The percentage of transferred CTLs in the tumor was assessed using flow cytometric analysis (n = 4 mice per group) (O). Tumor growth is indicated by luciferase activity in the lung (n = 5 mice per group) (P). The survival curve was monitored (n = 5 mice per group) (Q). Data are presented as means ± SEM and were analyzed by two‐tailed unpaired Student's t‐test (O, P) and Log‐rank test (Q). All results are representative of at least three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, no statistically significant.