Figure 4.
OTUD1 absence destabilizes Notch2 and restrains the Notch2 signaling in activated CD4+T cells. (A) Top 20 of KEGG pathway enriched for OTUD1-deficient CD4+ T cells stimulated with anti-CD3 (2 μg/mL) and anti-CD28 (0.4 μg/mL) antibodies compared with WT CD4+ T cells by the proteomics. (B) Heat map presenting the differential proteins that affect Th1 and Th17 differentiation according to KEGG analysis. (C) GSEA analysis of T-cell differential genes in OTUD1-deficient CD4+ T of aGVHD mice compared with WT CD4+ T cells shows JAK-STAT signaling pathway by sc-RNA seq. (D) CD4+ T cells isolated from WT or OTUD1−/− mice on B6 background were incubated with anti-CD3 (2 μg/mL) and anti-CD28 (0.4 μg/mL) antibodies and the protein levels of Notch2, Notch1, Notch3 and Notch4 were evaluated by western blot. (E) HEK293T cells were transfected with Myc-Notch2, control short hairpin RNA (shRNA) (shCON) or OTUD1shRNA (shOTUD1) plasmids. Myc-Notch2 proteins were immunoprecipitated with anti-Myc beads and the ubiquitination levels of Notch2 were evaluated by western blot. (F) Jurkat E6.1 cells were transfected with OTUD1-WT or OTUD1-AQ plasmids and incubated with anti-CD3 (0.1 μg/mL) antibodies and Notch2 proteins were immunoprecipitated with anti-Notch2 antibodies, the ubiquitination of Notch2 were analyzed by western blot. (G) Jurkat E6.1 cells were stimulated with anti-CD3 antibody (0.1 μg/mL) for 24 or 48 hours in vitro and the protein levels of OTUD1 and Notch2 were then assessed by western blot. (H) CD4+ T cells isolated from C57BL/6 mice were stimulated with anti-CD3 (2 μg/mL) and anti-CD28 (0.4 μg/mL) antibodies and the protein levels of Notch2 were evaluated by western blot. (I) CD4+ T cells isolated from WT or OTUD1−/− mice were stimulated with anti-CD3 (2 μg/mL) and anti-CD28 (0.4 μg/mL) antibodies for 24 hours and then the mRNA levels of Hes1, Hes5, Hey1 and HeyL were by RT-qPCR. (J) The mRNA levels of Hes1, Hes5, Hey1 and HeyL in PBMCs isolated from HCT patients (n = 104) were analyzed by RT-qPCR, the correlation between OTUD1 expression level and Hes1, Hes5, Hey1 and HeyL expression levels was analyzed. Data in panel I are represented as mean ± SD; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001 (2-tailed unpaired Student t test). Data in panels D-H are representative of 3 independent experiments and are summarized as mean ± SD of 3 experiments. HCT, hematopoietic cell transplantation.

OTUD1 absence destabilizes Notch2 and restrains the Notch2 signaling in activated CD4+T cells. (A) Top 20 of KEGG pathway enriched for OTUD1-deficient CD4+ T cells stimulated with anti-CD3 (2 μg/mL) and anti-CD28 (0.4 μg/mL) antibodies compared with WT CD4+ T cells by the proteomics. (B) Heat map presenting the differential proteins that affect Th1 and Th17 differentiation according to KEGG analysis. (C) GSEA analysis of T-cell differential genes in OTUD1-deficient CD4+ T of aGVHD mice compared with WT CD4+ T cells shows JAK-STAT signaling pathway by sc-RNA seq. (D) CD4+ T cells isolated from WT or OTUD1−/− mice on B6 background were incubated with anti-CD3 (2 μg/mL) and anti-CD28 (0.4 μg/mL) antibodies and the protein levels of Notch2, Notch1, Notch3 and Notch4 were evaluated by western blot. (E) HEK293T cells were transfected with Myc-Notch2, control short hairpin RNA (shRNA) (shCON) or OTUD1shRNA (shOTUD1) plasmids. Myc-Notch2 proteins were immunoprecipitated with anti-Myc beads and the ubiquitination levels of Notch2 were evaluated by western blot. (F) Jurkat E6.1 cells were transfected with OTUD1-WT or OTUD1-AQ plasmids and incubated with anti-CD3 (0.1 μg/mL) antibodies and Notch2 proteins were immunoprecipitated with anti-Notch2 antibodies, the ubiquitination of Notch2 were analyzed by western blot. (G) Jurkat E6.1 cells were stimulated with anti-CD3 antibody (0.1 μg/mL) for 24 or 48 hours in vitro and the protein levels of OTUD1 and Notch2 were then assessed by western blot. (H) CD4+ T cells isolated from C57BL/6 mice were stimulated with anti-CD3 (2 μg/mL) and anti-CD28 (0.4 μg/mL) antibodies and the protein levels of Notch2 were evaluated by western blot. (I) CD4+ T cells isolated from WT or OTUD1−/− mice were stimulated with anti-CD3 (2 μg/mL) and anti-CD28 (0.4 μg/mL) antibodies for 24 hours and then the mRNA levels of Hes1, Hes5, Hey1 and HeyL were by RT-qPCR. (J) The mRNA levels of Hes1, Hes5, Hey1 and HeyL in PBMCs isolated from HCT patients (n = 104) were analyzed by RT-qPCR, the correlation between OTUD1 expression level and Hes1, Hes5, Hey1 and HeyL expression levels was analyzed. Data in panel I are represented as mean ± SD; ∗P < .05, ∗∗P < .01, ∗∗∗P < .001 (2-tailed unpaired Student t test). Data in panels D-H are representative of 3 independent experiments and are summarized as mean ± SD of 3 experiments. HCT, hematopoietic cell transplantation.

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