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. 2017 Sep 4;214(9):2733–2758. doi: 10.1084/jem.20161903

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© 2017 Wiede et al.

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Figure 1. — PTPN2 deficiency influences thymocyte differentiation and increases STAT5 signaling. (A and B) Lineage (Lin)⁻ thymocytes from Ptpn2^+/+ (C57BL/6) and Ptpn2⁻^/⁻ (C57BL/6) mice (A) and poly (I:C)–treated Rosa26-eYFP;Ptpn2^fl/fl (C57BL/6) or Mx1-Cre;Rosa26-eYFP;Ptpn2^fl/fl (C57BL/6) mice (B) were stained with fluorochrome-conjugated antibodies against CD25, CD44, and c-KIT, and CD4/CD8 (DN) cell subsets were quantified by flow cytometry. (C) FACS-purified DN1 thymocytes from Ptpn2^+/+ (C57BL/6) and Ptpn2⁻^/⁻ (C57BL/6) mice were cultured for 10 d on OP9-DL1 stromal cells and stained for CD25, CD44, CD4, and CD8, and DN cell subsets were quantified by flow cytometry. (D, G, and H) Lin⁻ thymocytes from Ptpn2^+/+ (C57BL/6) and Ptpn2⁻^/⁻ (C57BL/6) mice were stained for CD25, CD44, and c-KIT and either intracellular p-(Y694) STAT5 (p-STAT5) (D), intracellular BCL-2 and MCL-1 (G), or the cell proliferation marker Ki67 (H) were determined in DN subsets by flow cytometry. (E) WBM cells from Ptpn2^+/+ (C57BL/6) and Ptpn2⁻^/− (C57BL/6) mice were cultured on OP9-DL1 stromal cells for 9 d. Lin⁻ thymocytes were harvested and stained for CD25, CD44, and intracellular p-(Y694) STAT5 (p-STAT5). The mean fluorescence intensities (MFIs) for intracellular staining of p-STAT5 was determined by flow cytometry. (F) FACS-purified Lin⁻c-KIT^hiSCA-1^hi BM cells from individual Ptpn2^+/+ (C57BL/6) (n = 5) and Ptpn2⁻^/⁻ (C57BL/6) (n = 5) mice were cultured on OP9-DL1 stromal cells for 9 d. DN2/3 thymocytes were harvested and stained for CD25 and CD44, and DN subsets were determined by flow cytometry. DN2/3 thymocyte lysates were resolved by SDS-PAGE and immunoblotted for p-STAT, STAT5, p-(Y1022/1023) JAK-1 (p-JAK1), JAK-1, PTPN2, and tubulin. Representative results (means ± SEM; Ptpn2^+/+, n = 4–7; Ptpn2⁻^/⁻, n = 4–9; Rosa26-eYFP;Ptpn2^fl/fl, n = 4; Mx1-Cre;Rosa26-eYFP;Ptpn2^fl/fl, n = 4) and representative cytometry profiles (C, D, and F) and immunoblots (F) from at least two independent experiments are shown. In F, each lane represents the cell lysate from one individual mouse. Significance was determined using two-tailed Mann-Whitney U test; *, P < 0.05; **, P < 0.01; ***, P < 0.001.