Extended Data Fig. 9: Effect of cytokines on regulating FBXO38 and PD-1 levels.

a–f, Naive CD8⁺ T cells were freshly isolated from the spleen and stimulated under different conditions. n = 3 biologically independent samples in all experiments. a, b, Cells were stimulated with anti-CD3 (2 μg ml⁻¹) alone without additional cytokines for 0–4 days. a, Fbxo38 transcriptional levels were measured by RT–qPCR. b, FBXO38 expression levels on day 4 were measured by immunoblotting. c, d, Cells were stimulated with anti-CD3/CD28 (2 μg ml⁻¹) in the presence or absence of 10 ng ml⁻¹ IL-10 or IL-15 for 0–4 days. e, f, Cells were stimulated with anti-CD3/CD28 (2 μg ml⁻¹) in the presence of 0–100 ng ml⁻¹ interleukins (IL-10 or IL-15) or interferons (IFNα, IFNβ or IFNγ) for four days. Naive cells were used as a control. g, The genetic regions to which STAT5 binds on the Fbxo38 gene loci are shown. This figure was reconstructed using the previously published GEO dataset GSM652878 and viewed with UCSC genome browser (genome version mm9)³⁰. h–n, Naive CD8⁺ T cells were freshly isolated from spleen and stimulated with anti-CD3/CD28 (2 μg ml⁻¹) in the presence of 0.5, 1, 10 or 100 ng ml⁻¹ IL-2 for 0–4 days. i–k, Percentages of PD-1⁺ and CD44⁺ of total CD8⁺ T cells and surface PD-1 MFI of CD8⁺ PD-1⁺ cells were assessed. l, m, Fbxo38 and Pdcd1 transcriptional levels of total CD8⁺ T cells were assessed by RT–qPCR and normalized to those of naive cells. n, FBXO38 expression was measured by immunoblotting. n = 3 biologically independent samples. Data are representative of four (a, c), three (d, e) or two (b, f, h–n) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, unpaired Student’s t-test with Welch’s correction (j (48 h, 0.5 versus 1), k (72 h, 0.5 versus 100, 96 h, 0.5 versus 1), l (48 h, 0.5 versus 1 and 0.5 versus 10, 96 h, 0.5 versus 10, 0.5 versus 100)), or  unpaired Student’s t-test. For gel source data, see Supplementary Fig. 1.

Source data