Fig 1: UA‐ERK1/2‐ULK1 cascade‐mediated autophagy regulates cellular metabolism and ROS levels to maintain T cell function. A,B) OT‐I CD8+ CTLs cells were treated with 10 µm UA or DMSO for 48 h. Metabolic intermediates were analyzed by mass spectrometry. Differential metabolites (A) and metabolic intermediates in the tricarboxylic acid (TCA) cycle (B) between DMSO‐ and UA‐treated CTLs are shown. Data are presented as means ± SEM (n = 3) and were analyzed by two‐tailed unpaired Student's t‐test (B). The data are from one experiment with three technical replicates per sample. C) 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. Oxygen consumption rate (OCR) was measured in treated CD8+ CTLs under basal and stimulated conditions with oligomycin, FCCP, and rotenone. Basal respiration, maximal respiration, spare respiratory capacity statistics, and ATP production were shown. Data are presented as means ± SEM (n = 3) and were analyzed by two‐tailed unpaired Student's t‐test. D) 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. Extracellular acidification rate (ECAR) of the treated OT‐I CTLs was measured under basal and stimulated conditions with glucose, rotenone, and 2‐deoxy‐glucose (2‐DG). Basal and maximal ECAR were shown. Data are presented as means ± SEM (n = 3) and were analyzed by two‐tailed unpaired Student's t‐test. E) Ratios of OCR to ECAR in DMSO‐ and UA‐treated CD8+ CTLs were shown. Data are presented as means ± SEM (n = 3) and were analyzed by two‐tailed unpaired Student's t‐test. F) OT‐I CD8+ CTLs were treated with 10 µm UA or DMSO for 48 h, followed by determination of TMRM staining 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 10 µm UA or DMSO for 48 h in the presence or absence of 10 µm chloroquine (CQ) for the last 4 hours, followed by flow cytometric analysis of ROS (DCFDA) (left) and mitochondrial superoxide (Mito‐sox) (right). Data are presented as means ± SEM (n = 3) and were analyzed by two‐tailed unpaired Student's t‐test. H) OT‐I CD8+ CTLs transduced with control, or ATG5 shRNA, were treated with 10 µm UA or DMSO for 48 h, followed by flow cytometric analysis of mitochondrial superoxide (Mito‐sox). Data are presented as means ± SEM (n = 3) and were analyzed by two‐tailed unpaired Student's t‐test. I) OT‐I CD8+ CTLs 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 flow cytometric analysis of ROS (DCFDA) and mitochondrial superoxide (Mito‐sox). Data are presented as means ± SEM (n = 3) and were analyzed by two‐tailed unpaired Student's t‐test. J) OT‐I CD8+ CTLs cells were treated with 10 µm UA or DMSO in the presence or absence of antimycin A (AA, 0.04 µm) or NAC (2.5 mm) for 48 h, followed by flow cytometric analysis of IFN‐γ and TNF‐α. Data 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, *** P < 0.001, and **** P < 0.0001; ns, no statistically significant.
Fig 2: 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.
Supplier Page from Abcam for Recombinant Human ULK1 protein