Fig 2: Gene deletion of Mkk4 and Mkk7 impairs differentiation process in immature neurons in GCL in Actin-CreERT2 (Cre+/−), Mkk4Δ/Δ, Mkk7Δ/Δ adult mice. (A). Double immunofluorescence against CR+ and DCX+ in WT adult mice (a,a’,a*), Actin-CreERT2(Cre−/−), Mkk4flox/flox, Mkk7flox/flox (b,b’,b*) and Actin-CreERT2(Cre+/−), Mkk4Δ/Δ, Mkk7Δ/Δ (c,c’,c*) adult mice. Merged channel is presented in (a–c), transformation in gray-inverted signal is represented for CR (a’–c’) and DCX (a*–c*). Arrowheads indicate double-positive cells (CR+/DCX+): The same cell is observed in the merged image and in the different fluorescence channels. Abbreviations. IML, inner molecular layer, GCL, granule cell layer, h, hilus, ML, molecular layer, SGZ, subgranule zone. Scale bar, 100 μm. (B). Graph represents neuronal counting of double-positive neurons (DCX+ and CR+) in the complete GCL of DG as a percent of total DCX+ neuronal count. A decrease is estimated in Actin-CreERT2(Cre+/−), Mkk4Δ/Δ, Mkk7Δ/Δ adult mice versus WT and Actin-CreERT2(Cre−/−), Mkk4flox/flox, Mkk7flox/flox adult mice. One-way ANOVA and post hoc Fisher’s Least Significant Difference (LSD) (n = 3) were used in the statistical analysis. **** p < 0.0001 vs. WT, #### p < 0.0001 vs. CRE (−).

Fig 3: The MKK4 and MKK7 pathway contribute to LPS-mediated inflammation. Rosa26-CreERT, Mkk4∆/∆, and Mkk7∆/∆ mice were treated with or without 20 mg/kg LPS by intraperitoneal injection (2 h). The amount of TNFα, IL6, IL1α, and IL1β in the serum was measured by ELISA (SE; n = 8). Statistically significant differences between wild-type and MLK-deficient mice are indicated. (mean ± S.E.M.; n = 8); (*) p < 0.05; (**) p < 0.01 vs. WT. All the experiments were repeated 3–4 times.

Fig 4: Effect of MKK4 or MKK7-deficiency on the response to LPS. BMDMs isolated from Rosa26-CreERT, Rosa26-CreERT-Mkk4-floxed, and Rosa26-CreERT-Mkk7-floxed conditional mice were treated with 4-hydroxytamoxifen for respective gene deletion. 5 days post deletion, BMDMs were treated with or without 100 ng/mL LPS for 10, 30, and 60 min. Protein extracts were examined by immunoblot analysis by probing with antibodies to MAP kinases and phospho-MAP kinases. All relative quantification values of western blots were measured in comparison to control. All the experiments were repeated 3–4 times.

Fig 5: Overexpression of constitutively active JNK promotes IBV-induced apoptosis a H1299 cells in duplicate were transfected with pcDNA-MKK7-JNK1 or pcDNA-MKK7-JNK1(APF), before being infected with IBV at MOI~2 or being mock infected. One set of cells were harvested for protein at the indicated time points and were subjected to Western blot analysis using the indicated antibodies. Beta-tubulin was included as loading control. Sizes of protein ladders in kDa were indicated on the left. Degree of JNK phosphorylation and the percentage of PARP cleavage was determined as in Fig. 3b. The experiment was repeated three times with similar results, and the result of one representative experiment is shown. b Huh-7 cells were transfected and infected similarly as in a. Western blot analysis and data quantification were performed as in a. The experiment was repeated three times with similar results, and the result of one representative experiment is shown.