Fig 1: Urolithin A regulates mouse CD8+ CTL function through the ERK pathway. A,B) OT‐I CD8+ CTLs were treated with DMSO or 10 µm UA for 48 h and stimulated with anti‐CD3/28 (1 + 3 µg ml−1) antibodies for 6 hours, followed by RNA‐seq analysis. A) Scatter plot showing the Gene Ontology (GO) Biological Process enrichment results in the differentially expressed genes (DEGs) with RNA sequencing data of DMSO‐ and 10 µm UA‐treated CTLs. The dot size indicates the relative number of differentially expressed genes contained in the GO terms, and the shade of the dots indicates the adjusted P value (Padj) of the enrichment. RNA‐seq data are from one experiment with three technical replicates per sample. B) Gene set enrichment analysis (GSEA) plot of differentially expressed genes in ERK1 and ERK2 cascade pathway between DMSO‐ and UA‐treated CTLs. RNA‐seq data are from one experiment with three technical replicates per sample. C) OT‐I CD8+ CTLs were treated with DMSO and 10 µm UA for 48 h and stimulated with anti‐CD3/CD28 (1 + 3 µg ml−1) antibodies for indicated time points, followed by immunoblots analysis. Representative immunoblot image (left) and quantification (right, normalized to total ERK1/2) of p‐ERK1/2 in DMSO‐ and UA‐treated CTLs. Data are presented as means ± SEM (n = 3) and were analyzed by two‐tailed unpaired Student's t‐test. The immunoblots were performed three times independently. D) CD8+ CTLs were treated with DMSO or 10 µm UA for 48 h. The fluorescent images of CD8+ CTLs stained by p‐ERK1/2 (red) (left). White scale bar = 2 µm. Quantification of relative p‐ERK1/2 fluorescence intensity (right). Data are presented as means ± SEM (n = 3) and were analyzed by two‐tailed unpaired Student's t‐test. The experiments were performed three times independently. E) CD8+ CTLs were treated with DMSO or UA (+, 5 µm; ++, 10 µm) for 48 h and fractionated. Then, the cytoplasmic and nuclear protein fractions were blotted for p‐ERK1/2, GAPDH (cytoplasmic marker), or Lamin B1 (nuclear marker). Quantification of p‐ERK1/2 in the nucleus and cytoplasm (right). Data are presented as means ± SEM (n = 3) and were analyzed by two‐tailed unpaired Student's t‐test. The immunoblots were performed three times independently. F) OT‐I CD8+ T cells were treated with DMSO or UA (10 µm) in the presence or absence of PD0325901 (PD, 10 µm) for 48 h, followed by anti‐CD3/28 stimulation for 6 h. Expression of IFN‐γ, TNF‐α, IL‐2, and Granzyme B (Gzm B) in CD8+ T cells 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. G) OT‐I CD8+ CTLs were treated with DMSO or UA (10 µm) in the presence or absence of PD0325901 (PD, 10 µm) for 48 h. Subsequently, cytotoxicity of the treated CD8+ CTLs against 10 nm OVA257‐264 peptide‐pulsed EL4 targets at indicated E:T ratios was determined in vitro. Data are presented as means ± SEM (n = 3) and were analyzed by two‐way ANOVA. H–K) OT‐I CTLs were treated with DMSO or UA (10 µm) in the presence or absence of PD0325901 (PD, 10 µm) for 48 h and transferred to B16‐MO5‐Fluc lung metastases‐bearing C57BL/6 mice on day 10 post‐inoculation. Schematic diagram of the adoptive transfer experiment (H). The percentage of transferred CTLs in the tumor was assessed using flow cytometric analysis (n = 4 mice per group) (I). Tumor growth is indicated by luciferase activity in the lung (n = 5 mice per group) (J). The survival curve was monitored (n = 5 mice per group) (K). Data are presented as means ± SEM and were analyzed by two‐tailed unpaired Student's t‐test (I, J) and Log‐rank test (K). All results are representative of at least three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, no statistically significant.
Fig 2: 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.
Fig 3: Urolithin A‐primed ERK1/2 triggers ULK1 activation and downstream autophagy flux to promote CD8+ T cell function. A) OT‐I CD8+ CTLs were treated with 10 µm UA or DMSO for 1, 4, 8, or 16 h, followed by immunoblot analysis. A representative image from three independent experiments is shown. B) OT‐I CD8+ CTLs were treated with 10 µm UA or DMSO for 48 h in the presence or absence of 10 µm chloroquine (CQ) for the last 4 h, followed by Western blot analysis. Torin2 (10 µm), an autophagy inducer, was used as a positive control. This experiment was repeated three times independently. C) Representative images of LC3I/II staining in OT‐I CD8+ T cells treated with 10 µm UA or DMSO for 48 h. The immunofluorescence images show LC3I/II (green) and DAPI (blue) in CTLs. White scale bars, 2 µm. This experiment was repeated three times independently. D) OT‐I CD8+ CTLs were treated with 10 µm UA or DMSO for 4, 8, and 16 h, followed by flow cytometric analysis of autophagosome. Representative flow cytometric histograms are shown (left). The mean fluorescence intensity (MFI) of autophagosome (right) 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. E) OT‐I CD8+ CTLs cells were treated with 10 µm UA or DMSO in the presence or absence of ERK inhibitor PD0325901 (PD, 10 µm) for 48 h, followed by Western blot analysis. Representative immunoblot image (left) and quantification of the indicated protein (right, normalized to β‐actin) 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. F) OT‐I CD8+ CTLs were treated with 10 µm UA or DMSO for 48 h in the presence or absence of ULK1 inhibitor MRT68921 (5 µm) for the last 4 h. Subsequently, cytotoxicity of the treated CD8+ CTLs against 10 nm OVA257‐264 peptide‐pulsed EL4 targets was determined in vitro at indicated E:T ratios. Data are presented as means ± SEM (n = 3) and were analyzed by two‐way ANOVA. G) OT‐I CD8+ CTLs were treated with 10 µm UA or DMSO for 48 h in the presence or absence of ULK1 inhibitor MRT68921 (5 µm) for the last 4 h and then stimulated with anti‐CD3/28 for 6 h. Production of IFN‐γ, TNF‐α, IL‐2, and granzyme B (Gzm 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. H) OT‐I CD8+ CTLs cells were treated with 10 µm UA or DMSO in the presence or absence of ERK inhibitor PD0325901 (PD, 10 µm) for 48 h, followed by Western blot analysis. 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. I) OT‐I CTLs were treated with 10 µm UA or DMSO for 48 h, followed by anti‐CD3/28 stimulation for 10 min. The co‐localization of phosphorylated ERK1/2 (p‐ERK1/2) and ULK1 was assessed by immunofluorescence staining analysis. Immunofluorescence images show p‐ERK (red), ULK1 (green), and DAPI (blue) in CTLs. White scale bars, 2 µm. This experiment was repeated three times independently. J) Co‐immunoprecipitation analysis of endogenous ERK1/2 and ULK1 using ERK1/2 antibody in OT‐I CTLs. This experiment was repeated three times independently. K) Purified ULK1 and ERK1 were incubated with MEK and ATP in the presence or absence of UA (10 µm) for in vitro kinase assay. The reactions were subjected to immunoblot analysis with indicated antibodies. Representative immunoblot image (left) and quantification of the serine phosphorylation of ULK1 (right, normalized to total ULK1) are shown. Quantification data from three independent experiments are presented as means ± SEM (n = 3) and were analyzed by two‐tailed unpaired Student's t‐test. L) Purified ULK1 and AMPKβ2 were incubated with ERK1 and ATP in the presence or absence of UA (10 µm) for in vitro kinase assay. The reactions were subjected to immunoblot analysis with indicated antibodies. Quantification of p‐AMPKβ2 (normalized to total AMPKβ2) from three independent experiments are presented as means ± SEM (n = 3) and were analyzed by two‐tailed unpaired Student's t‐test. All results are representative of at least three independent experiments. * P < 0.05, ** P < 0.01, and *** P < 0.001; ns, no statistically significant.
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