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HEP_25982_sm_SuppFig1.tif279KSupporting Figure 1. γδT cells in the spleen were not the main producer of IL-17A. Mice were injected i.p. with acetaminophen (APAP) at 400 mg/kg (body weight) or equal volumes of PBS as a control. The splenic leukocytes were isolated and the cell number was determined. (A and B) No alteration in the percentage and absolute number of γδT cells in the spleen. Leukocytes were isolated from the spleen 24 hours after APAP or PBS challenge. The percentage of total γδT cells among all leukocytes in the spleen were measured by flow cytometry and statistically analyzed (A). The absolute number of γδT cells in the spleen was calculated based on the total number of leukocytes and percentage (B). (C and D) No alteration in the IL-17A-producing γδ?T cells in the spleen. Leukocytes were isolated from the liver 24 hours after APAP or PBS challenge and stimulated with PMA and ionomycin for 4 hours in vitro. The percentage of IL-17A+ γδ?T cells was detected (C) and compared (D). (E) Few γδT cells in the spleen expressed IL-23 receptor. The leukocyte from the spleen and the liver of untreated C57BL/6 mice were stained by FITC-anti-CD3,PE-anti-IL23R,Percp-cy5.5-anti-CD45.2, and APC-anti-γδ?TCR. The γδ?T cells were gated firstly. (F) Most of γδT cells in the spleen expressed CD27. The leukocyte from the spleen and the liver of untreated C57BL/6 mice were stained by FITC-anti-CD27, PE-anti-CCR6, Percp-cy5.5-anti-CD3, and APC-anti-γδ?TCR. The data are representative of three independent experiments and are shown as the mean ± SEM.
HEP_25982_sm_SuppFig2.tif1733KSupporting Figure 2. γδT cells deficiency ameliorates liver inflammation and neutrophil infiltration. TCRδ-/- mice and the age-matched control mice were injected i.p. with APAP at 400 mg/kg (body weight). The ALT level, the survival ratio, and neutrophils in the liver were analyzed at 24 hours after APAP challenge. (A and B) Decreased liver injury in TCRδ-/- mice. ALT in the sera was measured (A).Liver specimens were excised and fixed in 4% paraformaldehyde to generate tissue sections stained with hematoxylin/eosin (B, H&E staining; original magnification, 50×). (C) Improved survival of TCRδ-/- mice. (D and E) Reduced neutrophil infiltration into the liver in TCRδ-/- mice. Leukocytes were isolated from the liver 24 hours after APAP challenge. The percentage of neutrophils was determined by flow cytometry (D, left panel) and was statistically analyzed (D, right panel). The absolute number of neutrophils was calculated and compared (E). The data are representative of three independent experiments and are shown as the mean ± SEM. *, P<0.05;**, P<0.01.
HEP_25982_sm_SuppFig3.tif1489KSupporting Figure 3. IL-23p40 deficiency ameliorates liver inflammation. IL-23p40-/- mice and the age-matched control mice were injected i.p. with APAP at 400 mg/kg (body weight). The ALT level and the survival ratio were analyzed at 24 hours after APAP challenge. (A and B) Decreased liver injury in IL-23p40-/- mice. ALT in the sera was measured (A).Liver specimens were excised and fixed in 4% paraformaldehyde to generate tissue sections stained with hematoxylin/eosin (B, H&E staining; original magnification, 50×). (C) Improved survival of IL-23p40-/- mice. The data are representative of three independent experiments and are shown as the mean ± SEM. **, P<0.01.
HEP_25982_sm_SuppFig4.tif1498KSupporting Figure 4. Inhibition of macrophages ameliorates liver inflammation. Mice were injected i.p. as indicated with APAP at 400 mg/kg (body weight) or equal volumes of PBS as a control. Mice were treated with GdCl3 at 20 mg/kg (body weight) at 24 hours before acetaminophen challenge. The sera were collected and the survival ratio was calculated 24 hours after APAP challenge. (A and B) Attenuated liver injury in mice treated with GdCl3. ALT in the sera was measured (A).Liver specimens were excised and fixed in 4% paraformaldehyde to generate tissue sections stained with hematoxylin/eosin (B,H&E staining; original magnification, 50×). (C) Improved survival of mice treated with GdCl3. The data are representative of three independent experiments and are shown as the mean ± SEM. **, P<0.01.

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