Fig 2: Pharmacological inhibition of ACK1 ameliorates mice with endotoxic shock. (A) C57BL/6 mice were injected AIM-100 (5, 10, 20 µg/g body weight) or vehicle for 2 hours followed by LPS challenge (37.5 µg/g of body weight), and the mortality of mice were observed (n = 8 mice/group). Survival curves were estimated by using Kaplan–Meier method and the Log-rank test was applied to determine the differences of survival rate. Statistically significant was assumed for P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and ns, not significant. (B–G) C57BL/6 mice were injected AIM-100 (5, 10, 20 µg/g body weight) or vehicle for 2 hours followed by LPS challenge (10 µg/g of body weight). ELISA measures the levels of TNF-a, IL-6 and IL-12 in serum at 3 hours (B). The lungs and livers of the endotoxic shock model mice were soaked in 4% paraformaldehyde and then paraffin embedded were stained with H&E at 12 hours (C). TUNEL analysis of apoptosis of lung and liver cells at 12 hours (C). The expression levels of CD40 and CD86 on splenic F4/80+ macrophages (D, E) and CD11c+ DCs (F, G) of endotoxin shock mice were detected by flow cytometry at 12 hours. The data are shown as the means ± SEM (n=6 mice/group) and are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, as determined by ANOVA tests; ns denotes p > 0.05.

Fig 3: ACK1 augments the activation of BMDMs induced by TLRs. (A–C) BMDMs were infected with lentivirus that highly expresses ACK1 (ACK1-LV) or negative control lentivirus (NC-LV) for 3 days followed by stimulation of LPS (100 ng/mL), R848 (1 µg/mL) and CpG-1826 (1 µM). The expression of CD86 (A) on BMDMs was detected by flow cytometry at 24 hours. The levels of TNF-a and IL-12 in culture supernatant were detected by ELISA at 24 hours (B). The phosphorylation levels of p38, Erk, JNK and p65 were analyzed by western blot at 1 hour (C). (D–F) BMDMs were infected with lentivirus that knockdown ACK1 expression (RNAi-ACK1-LV) or negative control lentivirus (RNAi-NC-LV) for 3 days followed by stimulation of LPS (100 ng/mL), R848 (1 µg/mL) and CpG-1826 (1 µM). The expression of CD86 (D) on BMDMs was detected by flow cytometry at 24 hours. The levels of TNF-a and IL-12 in culture supernatant were detected by ELISA at 24 hours (E). The phosphorylation levels of p38, Erk, JNK and p65 were analyzed by western blot at 1 hour (F). The data shown represent the means of three independent experiments and the error bars represent the S.E.M. *p < 0.05, **p < 0.01, and ***p < 0.001, as determined by ANOVA tests; ns denotes p > 0.05.

Fig 4: Pharmaceutical inhibition of PKM2 relieves the lupus-symptoms in MRL/lpr mice. (A) Representative images of marked splenomegaly in all groups of mice. (B) Weight of spleen in all groups of mice. (C) ELISA analysis of the anti-dsDNA antibody in serum from PKM2-IN- or vehicle-treated MRL/lpr mice. (D) H&E staining of the kidney sections from all groups of mice. (E, F) Immunofluorescence staining to detect IgG (E) and IgM (F) deposit in the kidney sections. (G–I) FACS analysis of CD86 expression on splenic macrophages (G), DCs (H) and B cells (I) in all groups of mice. The data are shown as the means ± SEM (n=6 mice/group) and are representative of three independent experiments. *p < 0.05, **p < 0.01, as determined by ANOVA tests or t-tests.

Fig 5: Pharmaceutical inhibition of PKM2 relieves LPS-induced endotoxin shock. (A) C57BL/6 mice were intraperitoneally injected with PKM2-IN (5, 10, 20 µg/g body weight) or vehicle for 2 hours followed by LPS challenge (37.5 µg/g body weight) and the mortalities of mice were observed (n=9 mice/group). (B–G) C57BL/6 mice were intraperitoneally injected with PKM2-IN (5, 10, 20 µg/g body weight) or vehicle for 2 hours followed by LPS challenge (10 µg/g body weight). Levels of IL-6, IL-12 and TNF-a in serum were determined by ELISA after 3 hours (B). Lung and liver sections were stained with H&E and apoptosis in the cells of lung and liver was analyzed by TUNEL (C). FACS analysis was performed to assess the expressions of CD86 and CD40 on surface of splenic F4/80+ macrophages (D, E) and CD11c+ DCs (F, G) after 12 hours. The data are shown as the means ± SEM (n=6 mice/group) and are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, as determined by ANOVA tests.