Targeting OTUD1/NICD axis by dapagliflozin impairs T-cell pathogenicity and attenuates the severity of aGVHD mice. (A) 3D map showing the spatial positions of the amino acid residues of the catalytic triad of OTUD1 and the site (white sphere) with high druggability predicted by the Sitemap. (B) HEK293T cells transfected with Flag-NICD, HA-ub, pCDH-OTUD1 plasmids were incubated with potential drugs (100 nM) as indicated for 24 hours, and then the protein levels of Flag-NICD were evaluated by western blot. (C) HEK293T cells transfected with Flag-NICD, HA-ub, pCDH-OTUD1 plasmids were incubated with potential drugs (100 nM) as indicated for 24 hours, and then Flag-NICD proteins were immunoprecipitated with anti-Flag beads and the ubiquitination of Flag-NICD was analyzed by western blot. (D) HEK293T cells transfected with Flag-NICD, HA-ub, pCDH-OTUD1 plasmids were incubated with or without dapagliflozin (100, 500 nM) for 24 hours, and then the protein level of Flag-NICD were analyzed by western blot. (E) Molecular docking diagram of dapagliflozin and OTUD1. Green represents the compound dapagliflozin, purple dashed lines represent π-π interactions, yellow dashed lines represent hydrogen bonds, and proteins are shown in white cartoons. (F) CD4+ T cells isolated from C57BL/6 mice were incubated with anti-CD3 (2 μg/mL) and anti-CD28 (0.4 μg/mL) antibodies and incubated with or without dapagliflozin (5 μM), and then the expression of IFN-γ and IL-2 were analyzed by flow cytometry. (G-I) BM cells (1 × 107 per mouse) and splenocytes (5 × 106 per mouse) isolated from C57BL/6 mice were transferred into lethally irradiated BALB/c mice intraperitoneally injected with Ctrl (n = 8), 1 mg/kg (n = 10) or 10 mg/kg (n = 7) dapagliflozin from day 1 to day 10 after transplantation. The survival (G) of aGVHD mice was observed twice 1 day, and aGVHD scores (H) were observed and recorded every 2 days. (I) Representative images were shown in intestine, lung, liver and skin of aGVHD mice by HE staining (scale bar, 100 μm; arrows, infiltrating lymphocytes; triangles, apoptotic epithelial cells). Log-rank (Mantel-Cox) test was used to analyze the survival curve (G). Data in panel F are represented as mean ± SD; ∗P < .05, ∗∗P < .01 (2-tailed unpaired Student t test). Data in panels B-D are representative of 3 independent experiments and data in panel D are summarized as mean ± SD of 3 experiments.
Figure 7.

Targeting OTUD1/NICD axis by dapagliflozin impairs T-cell pathogenicity and attenuates the severity of aGVHD mice. (A) 3D map showing the spatial positions of the amino acid residues of the catalytic triad of OTUD1 and the site (white sphere) with high druggability predicted by the Sitemap. (B) HEK293T cells transfected with Flag-NICD, HA-ub, pCDH-OTUD1 plasmids were incubated with potential drugs (100 nM) as indicated for 24 hours, and then the protein levels of Flag-NICD were evaluated by western blot. (C) HEK293T cells transfected with Flag-NICD, HA-ub, pCDH-OTUD1 plasmids were incubated with potential drugs (100 nM) as indicated for 24 hours, and then Flag-NICD proteins were immunoprecipitated with anti-Flag beads and the ubiquitination of Flag-NICD was analyzed by western blot. (D) HEK293T cells transfected with Flag-NICD, HA-ub, pCDH-OTUD1 plasmids were incubated with or without dapagliflozin (100, 500 nM) for 24 hours, and then the protein level of Flag-NICD were analyzed by western blot. (E) Molecular docking diagram of dapagliflozin and OTUD1. Green represents the compound dapagliflozin, purple dashed lines represent π-π interactions, yellow dashed lines represent hydrogen bonds, and proteins are shown in white cartoons. (F) CD4+ T cells isolated from C57BL/6 mice were incubated with anti-CD3 (2 μg/mL) and anti-CD28 (0.4 μg/mL) antibodies and incubated with or without dapagliflozin (5 μM), and then the expression of IFN-γ and IL-2 were analyzed by flow cytometry. (G-I) BM cells (1 × 107 per mouse) and splenocytes (5 × 106 per mouse) isolated from C57BL/6 mice were transferred into lethally irradiated BALB/c mice intraperitoneally injected with Ctrl (n = 8), 1 mg/kg (n = 10) or 10 mg/kg (n = 7) dapagliflozin from day 1 to day 10 after transplantation. The survival (G) of aGVHD mice was observed twice 1 day, and aGVHD scores (H) were observed and recorded every 2 days. (I) Representative images were shown in intestine, lung, liver and skin of aGVHD mice by HE staining (scale bar, 100 μm; arrows, infiltrating lymphocytes; triangles, apoptotic epithelial cells). Log-rank (Mantel-Cox) test was used to analyze the survival curve (G). Data in panel F are represented as mean ± SD; ∗P < .05, ∗∗P < .01 (2-tailed unpaired Student t test). Data in panels B-D are representative of 3 independent experiments and data in panel D are summarized as mean ± SD of 3 experiments.

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