Fig 2: JNK2 deficiency augments lung inflammation and injury in CLP-induced sepsis. (A) Sectional lung histology of sham- or CLP-treated wild-type and JNK2 KO mice. (B) mRNA expressions of MCP-1 and KC from whole lung homogenates of sham-treated wild type mice or CLP-treated wild-type and JNK2 KO mice. N=3. Data are presented as means ± sem. ***p < 0.001; ****p < 0.0001.

Fig 3: Loss of JNK2 worsens lung inflammation and injury during pseudomonas pneumonia. Wild-type and JNK2 KO mice were subjected to i.n. pseudomonas aeruginosa (strain PA103)-induced pneumonia. (A) Mortality of PA103-treated wild-type and JNK2 KO mice. N=9-10. (B–E) Cell count and protein content (B) and neutrophil count (C) in BAL fluid, and mRNA expressions of inflammatory cytokines from whole lung homogenates (D), or sectional lung histology (E) from PBS- or PA103-treated mice. In (B), PBS: N=3-8; PA103: N=13-14. In (C), N=3-4. In (D), PBS: N=3; PA103: N=4-5. Data are presented as means ± sem. *p < 0.05; **p < 0.01.

Fig 4: JNK knockdown increase Puma mRNA expression.INS1 cells stably expressing shRNA directed against JNK1, JNK2, JNK3 or the non-sense control were exposed to 0.5 mM palmitate and 25 mM glucose for 12 or16 h (+). Relative mRNA expression was measured using quantitative RT-PCR and normalized to Hprt1. Relative DP5 mRNA expression at A: 12 h, B: 16 h. Relative Puma mRNA expression C: 12 h, D: 16 h. Data are shown with+SEM of five - six independent experiments. INS1 cells stably expressing shRNA directed against JNK1, JNK2, JNK3 or the non-sense control were exposed to 0.5 mM palmitate and 25 mM glucose (black bars) or vehicle (white bars) for 24 h. Relative Ins1 mRNA expression E: 24 h, Relative Ins2 mRNA expression, F: 24 h. G: Total insulin content (ng insulin/total DNA) after GSIS. Data are shown with+SEM of four independent experiments *P<0.05, **P<0.01, ***P<0.001.

Fig 5: Small RNA sequencing revealed that miR-221-5p is upregulated in response to LPS in vitro, resulting in JNK2 downregulation. mRNA expressions of JNK2 in LPS- (A) or PA103-treated (B) RAW 264.7 cells, or LPS-treated THP-1 cells (PMA-differentiated) (C) as indicated. (D) Heatmap of differential expressed miRNAs in RAW 264.7 cells treated with LPS for 0, 4, and 8h. (E) Seed sequence of miR-221-5p matching to JNK2 mRNA 3’UTR. (F) Levels of miR-221-5p in RAW 264.7 cells treated with LPS for different times. (G) JNK2 mRNA levels in mouse lung epithelial cells (MLE-12) treated with mimic control or miR-221-5p mimic (left panel), or inhibitor control or miR-221-5p inhibitor (right panel). (H) JNK2 mRNA levels in whole lung homogenates of mice intratracheally treated without or with miRNA inhibitor control or miR-221-5p inhibitor. (I) JNK2 3’UTR luciferase activity in the presence of miR-221-5p mimic (left panel; normalized to control mimic) or miR-221-5p inhibitor (right panel; normalized to control inhibitor). (J) JNK2 mRNA in the RNA-protein complex using Ago2 or control IgG for IP in the presence of miR-221-5p mimic (normalized to input). Data are presented as means ± sem. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns, not significant.