Extended Data Fig. 2: Proline deficiency does not alter NKp46+ ILC3 and other non-ILC3 immune cells.
From: Proline uptake promotes activation of lymphoid tissue inducer cells to maintain gut homeostasis
a, Comparison of intestinal NK/ILC1, ILC2 and ILC3 in mice fed by normal diet (ND) and proline free diet (PFD) (n = 5 per group; ns; ns; ns). b, Comparison of different ILC3 subgroups, including LTi, NKp46+ ILC3, NKp46−T-bet+ ILC3, NKp46−T-bet−CCR6− ILC3, in mice fed by ND and PFD (n = 5 per group; ns, ns, ns, ns). c-g, Comparison of CD11b+ cells (c), CD11b+Gr-1+ cells (d), T cells (e), CD4+ T cells, Th1, Th17, Treg (f), and B cells (g), in mice fed by ND and PFD (n = 4 per group; ns; ns; ns; ns; ns, ns, ns; ns). h, Flow cytometric analysis of IL-22 production by intestinal ILC3 (live lineage−RORγt+) in mice fed by ND or PFD. Percentages of IL-22+ ILC3 and their IL-22 MFI were calculated in the right (n = 7 per group; **p = 0.0048; **p = 0.0082). i, Flow cytometric analysis of IL-22 production by intestinal NKp46+ ILC3 in mice fed by ND or PFD. Percentages of IL-22+ NKp46+ ILC3 and their IL-22 MFI were calculated in the right (n = 7 per group; ns; ns). j, Flow cytometric analysis of IL-22 production by intestinal NKp46+ ILC3 of Rag2-/- mice fed by ND or PFD. Percentages of IL-22+ NKp46+ ILC3 and their IL-22 MFI were calculated in the right (n = 4 per group; ns; ns). k, The structural formula of proline analog. l, Intestinal LTi, NKp46+ ILC3 and ILC2 cells were sort-purified and cultured in PFM with or without the proline analog for 12 hours and then the cellular proteins incorporating the proline analog were detected by click reaction through adding azide coupled with biotin and PE streptavidin in sequence for reaction. The PE fluorescence intensity was tested by flow cytometry. m, The relative PE fluorescence intensity were calculated and the cells cultured without the proline analog was used as control (n = 4 per group; *p = 0.0339; **p = 0.0072). Data were representative of at least three independent experiments (a-j, l-m). Data were presented as the mean ± s.e.m, and statistical significance was determined by two-sided unpaired t-test (a-j, m). *p < 0.05; **p < 0.01; ns, not significant.
