Figure 4.
IL-25 and IFN-γ regulate Xbp1 mRNA splicing in ILC2s through p38-mediated IRE1α phosphorylation. (A–C) Sorted ILC2s (Lin−CD127+KLRG1+) were cultured in complete IMDM containing IL-2, IL-7, and IL-25 (10 ng/ml each) for 5 days, followed by treatment with CHX at the indicated concentrations for 2 days. Areg, IL-5, and IL-13 expressions in ILC2s (CD45.2+Lin−GATA3+) were analyzed by flow cytometry (A). Percentages of Areg+, IL-5+, and IL-13+ cells in ILC2s (B). Xbp1 mRNA splicing of ILC2s was detected by qRT-PCR (C). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (D–G) Sorted ILC2s (Lin−CD127+KLRG1+) were stimulated with the inhibitors of IL-25 downstream signaling pathways for 16 h, including JNK-IN-8 (10 μM), SCH772984 (1 μM), PH797804 (10 μM), and andrographolide (10 μM) as shown in D. Xbp1 mRNA splicing of ILC2s was detected by qRT-PCR (E). Xbp1s expression in ILC2s (CD45.2+Lin−GATA3+) was analyzed by flow cytometry (F). MFI of Xbp1s in ILC2s (G). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 5 per group). (H and I) Sorted ILC2s (Lin−CD127+KLRG1+) were stimulated with PH797804 (10 μM) for 16 h in complete IMDM containing IL-2, IL-7, and IL-25 (10 ng/ml each). Analysis of Areg, IL-5, and IL-13 expression in ILC2s (CD45.2+Lin−GATA3+) by flow cytometry (H). Percentages of Areg+, IL-5+, and IL-13+ cells in ILC2s (I). Data were compiled from three independent experiments. Data are shown as the mean ± SD (n = 5 per group). (J) Sorted ILC2s (Lin−CD127+KLRG1+) were cultured for 5 days, followed by PH797804 treatment at 10 μM for 16 h. The level of p-IRE1α and Xbp1s in ILC2s was detected by western blotting. β-Actin served as the internal control. Data are representative of three independent experiments. (K) Sorted ILC2s (Lin−CD127+KLRG1+) were cultured for 5 days in the presence of IL-2, IL-7, and IL-25 (10 ng/ml each), and IL-25 was removed for 2 days, followed by restimulation with or without IL-25 (10 ng/ml) for 1 day. The level of p-p38, p-IRE1α, and Xbp1s in ILC2s was analyzed by western blotting. β-Actin served as the internal control. Data are representative of three independent experiments. (L) Sorted ILC2s (Lin−CD127+KLRG1+) were stimulated with the IFN-γ (500 ng/ml) for 3 days in complete IMDM containing IL-2, IL-7, and IL-25 (10 ng/ml each). Expression of p-p38, p-IRE1α, and Xbp1s in ILC2s was analyzed by western blotting. β-Actin served as the internal control. Data are representative of three independent experiments. **P < 0.01, ***P < 0.001. Source data are available for this figure: SourceData F4. Refer to the image caption for details.

IL-25 and IFN-γ regulate Xbp1 mRNA splicing in ILC2s through p38-mediated IRE1α phosphorylation. (A–C) Sorted ILC2s (Lin−CD127+KLRG1+) were cultured in complete IMDM containing IL-2, IL-7, and IL-25 (10 ng/ml each) for 5 days, followed by treatment with CHX at the indicated concentrations for 2 days. Areg, IL-5, and IL-13 expressions in ILC2s (CD45.2+Lin−GATA3+) were analyzed by flow cytometry (A). Percentages of Areg+, IL-5+, and IL-13+ cells in ILC2s (B). Xbp1 mRNA splicing of ILC2s was detected by qRT-PCR (C). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 6 per group). (D–G) Sorted ILC2s (Lin−CD127+KLRG1+) were stimulated with the inhibitors of IL-25 downstream signaling pathways for 16 h, including JNK-IN-8 (10 μM), SCH772984 (1 μM), PH797804 (10 μM), and andrographolide (10 μM) as shown in D. Xbp1 mRNA splicing of ILC2s was detected by qRT-PCR (E). Xbp1s expression in ILC2s (CD45.2+Lin−GATA3+) was analyzed by flow cytometry (F). MFI of Xbp1s in ILC2s (G). Data were compiled from two independent experiments. Data are shown as the mean ± SD (n = 5 per group). (H and I) Sorted ILC2s (Lin−CD127+KLRG1+) were stimulated with PH797804 (10 μM) for 16 h in complete IMDM containing IL-2, IL-7, and IL-25 (10 ng/ml each). Analysis of Areg, IL-5, and IL-13 expression in ILC2s (CD45.2+Lin−GATA3+) by flow cytometry (H). Percentages of Areg+, IL-5+, and IL-13+ cells in ILC2s (I). Data were compiled from three independent experiments. Data are shown as the mean ± SD (n = 5 per group). (J) Sorted ILC2s (Lin−CD127+KLRG1+) were cultured for 5 days, followed by PH797804 treatment at 10 μM for 16 h. The level of p-IRE1α and Xbp1s in ILC2s was detected by western blotting. β-Actin served as the internal control. Data are representative of three independent experiments. (K) Sorted ILC2s (Lin−CD127+KLRG1+) were cultured for 5 days in the presence of IL-2, IL-7, and IL-25 (10 ng/ml each), and IL-25 was removed for 2 days, followed by restimulation with or without IL-25 (10 ng/ml) for 1 day. The level of p-p38, p-IRE1α, and Xbp1s in ILC2s was analyzed by western blotting. β-Actin served as the internal control. Data are representative of three independent experiments. (L) Sorted ILC2s (Lin−CD127+KLRG1+) were stimulated with the IFN-γ (500 ng/ml) for 3 days in complete IMDM containing IL-2, IL-7, and IL-25 (10 ng/ml each). Expression of p-p38, p-IRE1α, and Xbp1s in ILC2s was analyzed by western blotting. β-Actin served as the internal control. Data are representative of three independent experiments. **P < 0.01, ***P < 0.001. Source data are available for this figure: SourceData F4.

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