Fig 1.
Con A-induced hepatitis in STAP-1 KO and Tg mice.
(A) STAP-1 expression in thymus and spleen from C57BL/6 mouse, 2E10 (mouse NKT cells), NIH-3T3 (mouse fibroblasts) and Ba/F3 (pro-B cells) was detected by western blotting. Actin was detected as a loading control. (B) STAP-1 expression in spleens of C57BL/6 (WT), STAP-1 KO (S1KO) and STAP-1 Tg (S1Tg) mice was detected by western blotting. Actin was detected as a loading control. (C) WT, S1KO and S1Tg mice were intravenously inject with Con A (10 mg/kg). After Con A injection, mice were sacrificed, and liver tissues were collected. The necrotic areas in livers of WT, S1KO and S1Tg mice at 12 h after intravenous injection of Con A was visualized by H&E staining. Representative images of H&E staining from four independent experiments with a total 12–14 mice are shown. Magnification; Objective x 4, Optical x 12, Scale bar = 200 μm. (D) Necrotic area percentages in livers of WT, S1KO and S1Tg mice at 12 h after intravenous injection of Con A were measured by ImageJ. Four independent experiments were performed and pooled data were shown mean + SEM (WT n = 14, S1KO n = 13, S1Tg n = 12). (E) Liver tissues were collected from WT, S1KO and S1Tg mice before and after intravenous Con A injection (12 or 24 h), cleaved caspase-3 in the livers was detected by western blotting. Actin was detected as a loading control. (F) Plasma ALT levels of WT, S1KO and S1Tg mice at 12 h after intravenous injection of Con A were measured. Four independent experiments were performed and pooled data were shown mean + SEM (WT n = 15, S1KO n = 14, S1Tg n = 12). (G, H) Plasma IL-4 and IFN-γ levels of WT, S1KO and S1Tg mice at 3 and 12 h after intravenous injection of Con A were measured by ELISA. Three independent experiments were performed and pooled data were shown mean + SEM (WT n = 11, S1KO n = 10, S1Tg n = 9). *; p<0.05, ***; p<0.001 by Mann-Whitney U-test. ns = no significance.
Fig 2.
α-GalCer-induced hepatitis in STAP-1 KO and Tg mice.
(A) The necrotic areas in livers of C57BL/6 (WT), S1KO and S1Tg mice at 16 h after intravenous injection of α-GalCer (0.1 mg/kg) was visualized by H&E staining and percentages of the area were measured by ImageJ. Three independent experiments were performed and pooled data were shown mean + SEM (WT n = 11, S1KO n = 11, S1Tg n = 10). (B) Plasma ALT levels of WT, S1KO and S1Tg mice at 16 h after intravenous injection of α-GalCer were measured. Three independent experiments were performed and pooled data were shown mean + SEM (WT n = 14, S1KO n = 14, S1Tg n = 12). (C, D) Plasma IL-4 and IFN-γ levels of WT, S1KO and S1Tg mice at 3 and 16 h after intravenous injection of α-GalCer were measured by ELISA. Three independent experiments were performed and pooled data were shown mean + SEM (WT n = 14, S1KO n = 13–14, S1Tg n = 11–13). *; p<0.05, ***; p<0.001 by Mann-Whitney U-test. ns = no significance.
Fig 3.
iNKT cell population in STAP-1 KO and Tg mice.
(A) iNKT cells were purified from spleen and STAP-1 expression was analyzed by western blotting. Actin was detected as a loading control. (B) iNKT cell (CD1d dimer+/TCRβ+) and T cell (CD1d dimer-/TCRβ+) populations in livers of C57BL/6 (WT), S1KO and S1Tg mice were analyzed by flowcytometric analysis. Upper panels show the representative data of flowcytometric analysis. Lower graphs are summarized data of the flowcytometric analysis. Three independent experiments were performed and pooled data were shown mean + SEM (WT n = 7–8, S1KO n = 9, S1Tg n = 9). (C) iNKT cell (CD1d dimer+/TCRβ+) and T cell (CD1d dimer-/TCRβ+) populations in spleens of WT, S1KO and S1Tg mice were analyzed by flowcytometric analysis. Upper panels show the representative data of flowcytometric analysis. Lower graphs are summarized data of the flowcytometric analysis. Three independent experiments were performed and pooled data were shown mean + SEM (WT n = 7–8, S1KO n = 9, S1Tg n = 9). (D) Analysis of iNKT cell development in thymuses of WT, S1KO and S1Tg mice was performed by flowcytometric analysis. Four figures are summarized graphs of iNKT cell stage 0 to 3. Data were shown mean + SEM of single experiment (WT n = 3, S1KO n = 3, S1Tg n = 3). *; p<0.05, **; p<0.01, ***; p<0.001 by Mann-Whitney U-test. ns = no significance.
Fig 4.
Reduction of cytokine production in STAP-1 2E10 cells.
(A) Expression of endogenous and exogenous STAP-1 in Mock and STAP-1 2E10 cells was detected by western blotting. Arrowhead and asterisk are shown endogenous and exogenous STAP-1, respectively. Actin was detected as a loading control. (B, C) Mock and STAP-1 2E10 cells were stimulated with Con A (0, 1 and 10 μg/ml) for 3 h, and IL-4 and IFN-γ mRNA levels were measured by qPCR. Data were normalized by a ΔΔCt method using GAPDH expression levels. Result shown was representative of two independent experiments. Data was shown mean + SEM. (D, E) Mock and STAP-1 2E10 cells were stimulated with Con A (0 and 50 μg/ml) for 24 h, and IL-4 and IFN-γ protein levels in supernatants were measured by ELISA. Data were pooled from 3 independent experiments and shown mean + SEM. *; p<0.05, **; p<0.01 by Mann-Whitney U-test. (F) Mock and STAP-1 2E10 cells were stimulated with Con A (10 μg/ml) for indicated times, and phosphorylation of Akt and MAPKs was analyzed by western blotting. Data shown are representative of three independent experiments. Arrowhead and asterisk are shown endogenous and exogenous STAP-1, respectively. Actin was detected as a loading control.