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Journal: Frontiers in Immunology
Article Title: Cytosolic protein translation regulates cell asymmetry and function in early TCR activation of human CD8 + T lymphocytes
doi: 10.3389/fimmu.2024.1411957
Figure Lengend Snippet: Cytosolic protein translation supports tubulin cytoskeleton reorganisation in human CD8 + cytotoxic T lymphocytes during T-cell receptor (TCR) and CD28 activation. (A, B) Fluorescence images of human cytotoxic T lymphocytes (CTLs) treated with vehicle (CTRL), cycloheximide (CHX; 20 μg/mL), or puromycin (PURO; 50 μg/mL) for 1 h and conjugated with (A) control or (B) stimulating beads. Green, α-tubulin; magenta, PCM1; blue, DAPI; BF, brightfield. Maximal projections are shown. Bar, 5 μm. (C) Graph showing quantification of microtubule-organising centre (MTOC) distance to the immunological synapse (IS). Data are mean ± SD; CTRL(−), n = 74; CTRL(+), n = 85; CHX(−), n = 74; CHX(+), n = 90; PURO(−), n = 74; and PURO(+), n = 80, cells analysed from six healthy donors; one-way ANOVA. **, p < 0.01; ****, p < 0.0001; ns, non-significant. (D) K40-α-tubulin acetylation in CTLs pre-treated as in panel A were stimulated or not with αCD3/αCD28 tetramers at indicated times. (E) Graph showing densitometric quantification of acetylated K40-α-tubulin normalised to total α-tubulin and referenced to non-stimulated CTRL. Data are mean ± SEM; n = 3; two-way ANOVA. *, p < 0.05. (F) Phosphorylation of PLCγ1 (pY783) and Erk1/2 (pT202/Y204) in CTLs pre-treated as in panel A and stimulated or not with αCD3/αCD28 tetramers at indicated times. (G, H) Graphs showing quantification of densitometries of (G) PLCγ1 (pY783) and (H) Erk1/2 (pT202/Y204) normalised to PLCγ1 and Erk1/2 and referenced to non-stimulated CTRL. Data are mean ± SEM; (G) , n = 7; (H) , n = 4; two-way ANOVA. *, p < 0.05; **, p < 0.01; ****, p < 0.0001. See also Supplementary Figure 1 .
Article Snippet: Antibodies used include pS2448 mTOR, mTOR, pS235/S236 S6, S6 ribosomal subunit, pS473 Akt, Akt, pY783 PLCγ1, PLCγ1, pS402/Y404 Erk1/2, Erk1/2, and PCM1 (all 1:1,000 WB, 1:100 IF; Cell Signaling Technology, Beverly, MA, USA).
Techniques: Activation Assay, Fluorescence, Control, Phospho-proteomics
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