Fig 2: TRIM11‐regulated apoptosis in AC16 cells probably by modulating DUSP1 expression (a, b). After injection with the oeDUSP1 lentivirus, the mRNA (a) and protein (b) expression of DUSP1 were detected. ***p < .001 versus vector. AC16 cells were injected with the oeTRIM11 or oeDUSP1 lentivirus. (c) The protein expressions of cleaved caspase‐3, Bcl‐2, Bax, JNK1/2, p‐JNK1/2 were detected. (d) Apoptosis at 48 h was detected. ***p < .001 versus vector; ## p < .01 versus oeTRIM11 + vector. Control, AC16 cells; vector, negative control of DUSP1 overexpression

Fig 3: Ninj1 deficiency attenuated IR-induced intrahepatic inflammation and neutrophil infiltration by DUSP1-dependent inhibition of p38 and JNK pathways in vivo. WT→WT and KO→WT mice were treated with either DUSP1-siRNA or NS-siRNA before the onset of liver ischemia, as described in the Materials and Methods section. The liver tissues and blood specimens were collected at 24 hours after reperfusion. (A) Western blot analysis of DUSP1, p-P38, T-p38, p-JNK, T-JNK, and β-actin expression. Relative intensity was analyzed by ImageJ software (National Institutes of Health). (B) Quantitative RT-PCR analysis of liver inflammatory cytokine and chemokine gene induction (IL6, IL1β, TNF-α, and CXCL-1). (C) Enzyme-linked immunosorbent assay (ELISA) of inflammatory cytokine and chemokine levels in serum. All results are representative of at least 2 independent experiments. (D and E) Representative immunofluorescence staining of LY6G (red) and 4’,6-diamidino-2-phenylindole dihydrochloride (DAPI) (blue) in livers from each group, quantification of LY6G+ cells per high-power field (HPF), Scale bar: 100 μm; n = 6 mice/group. ∗P < .05. All data are presented as the means ± SD. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. T-p38, Total-p38; T-JNK, Total-JNK; LY6G, lymphocyte antigen 6 complex locus g.

Fig 4: Ninj1 deficiency attenuated liver IR injury in a DUSP1-dependent manner. WT→WT and KO→WT mice were treated with either DUSP1-siRNA or NS-siRNA before the onset of liver ischemia, as described in the Materials and Methods section. The liver tissues and blood specimens were collected at 24 hours after reperfusion. (A) Representative H&E staining of ischemic livers from each group for different times. Scale bar: 100 μm. (B) Necrotic areas in ischemic livers analyzed by ImageJ software (National Institutes of Health). (C) Suzuki histologic grading of IR injury. (D and E) Serum levels of ALT and AST. (F) Immunofluorescence staining of TUNEL+ cells in ischemic livers from each group. Scale bar: 100 μm. (G) Quantification of TUNEL+ cells per high-power field. (H) Western blot analysis of Bcl-2, Bax, C-caspase-3, and β-actin expression in livers from each group. Relative intensity was analyzed by ImageJ software. All results are representative of at least 2 independent experiments; n = 6 mice/group. ∗P < .05. All data are presented as the means ± SD. C-caspase-3, Cleaved-caspase-3.

Fig 5: Ninj1 deficiency diminished the inflammatory response in KCs through DUSP1-dependent deactivation of JNK and p38 in vitro. KCs isolated from WT and Ninj1 KO mice were stimulated with LPS for 6 hours. The cells and culture supernatant were collected. (A) Western blot analysis of MyD88 and β-actin expression. Relative intensity analyzed by ImageJ software (National Institutes of Health). (B) Western blot analysis of p-P38, T-p38, p-JNK, T-JNK, p-ERK, T-ERK, and β-actin expression. Relative intensity analyzed by ImageJ software. (C) Western blot analysis of Ninj1, p-ASK1, p-TAK1, DUSP1, and β-actin expression. Relative intensity analyzed by ImageJ software. WT and KO KCs were transfected transiently with DUSP1-siRNA or NS-siRNA followed by LPS stimulation for 6 hours. The cells and culture supernatant were collected. (D) Western blot analysis of DUSP1, p-P38, T-p38, p-JNK, T-JNK, and β-actin expression. Relative intensity analyzed by ImageJ software. (E) Quantitative RT-PCR analysis of KC inflammatory cytokine and chemokine gene induction (IL6, IL1β, TNF-α, and CXCL-1). (F) Enzyme-linked immunosorbent assay of inflammatory cytokine and chemokine levels in cell supernatant. All results are representative of at least 2 independent experiments. ∗P < .05. All data are presented as the means ± SD. ELISA, enzyme-linked immunosorbent assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PBS, phosphate-buffered saline. T-p38, Total-p38; T-JNK, Total-JNK; T-ERK, Total ERK.