Fig 2: Model of IKK2, TAK1 and TPL2 functions in LMP1-CTAR2 signaling.IKK2 acts as critical mediator of both JNK and canonical NF-?B activation by CTAR2. TPL2 transmits JNK activation signals downstream of IKK2. The TAK1 protein and NEMO are required for IKK2 recruitment to the LMP1 signalosome, thereby mediating activation of IKK2 and its downstream effectors I?Ba/NF-?B and JNK. TAK1 kinase activity is dispensable for IKK2 and NF-?B activation, but has a role in JNK activation parallel to IKK2. KD, TAK1 kinase domain.

Fig 3: Essential functions of IKK2 and NEMO in JNK activation by LMP1.a LMP1 fails to induce JNK after the knockout of IKK2 in MEFs. LMP1 activity was induced by antibody crosslinking and JNK activation was monitored by immunoblotting. b Pharmacological inhibition of IKK activity interferes with JNK activation by LMP1. NGFR-LMP1 was induced in the presence of solvent (DMSO) or 5 µM of the IKK inhibitor VIII (ACHP). c, d IKK2 has a unique role in LMP1 signaling as compared to the TNFa and IL-1 pathways. Wildtype and IKK2-/-MEFs were stimulated with 20 ng/ml TNFa (c) or 10 ng/ml IL-1 (d) for the indicated times. e IKK1 is dispensable for JNK activation by LMP1. IKK1 was targeted by CRISPR/Cas9 in wildtype MEF:NGFR-LMP1wt and IKK2-/-MEF:NGFR-LMP1wt cells. LMP1 activity was induced in non-targeted, IKK1crKOMEF:NGFR-LMP1wt (clone 69) and IKK1crKOIKK2-/-MEF:NGFR-LMP1wt (clone 8) cells. Activation of JNK and canonical NF-?B was monitored. f The knockout of NEMO causes a defect in JNK activation by LMP1. NEMO was inactivated in wtMEF:NGFR-LMP1wt cells by CRISPR/Cas9 technology. NGFR-LMP1 activity was induced in non-targeted cells and the NEMOcrKO clones 2, 7, and 19. JNK and canonical NF-?B were analysed. CRISPR/Cas9 knockouts were verified by immunoblotting (this Figure) and sequencing (see Supplementary Table 2). a–f The data are representative of at least two independent experiments. For immunoblot quantification and statistics see Supplementary Table 3.

Fig 4: The JNK pathway is implicated in the atrial fibrosis. After the fibroblasts were treated with JNK inhibitor SP600125, the cell viability was measured by CCK-8 assay (A), the proliferation ability was detected by BrdU assay (B), cell apoptosis was evaluated by TUNEL staining (C), and cell migration ability was examined by cell scratch assay (D). Then, qRT-PCR and Western blot were used to assess the mRNA and protein expressions of collagen I and a-SMA (E,F); *P < 0.05, **P < 0.01.

Fig 5: Effects of honokiol on the MAPK pathway in TGF-ß1-stimulated fibroblasts. (A) The effects of honokiol on the MAPK signalling pathway were assessed by western blot analysis to detect the expression of phosphorylated ERK, JNK and p38. The cell lysates were prepared from fibroblasts pre-treated with honokiol for 2 h and subsequently induced by TGF-ß1 for 1 h. (B-D) Bar diagrams of p-ERK, p-JNK andp-p38 relative to total ERK, JNK, and p38, respectively. Data are presented as the means ± SEM, **P<0.01, vs. the control group. #P<0.05, ##P<0.01 compared with the TGF-ß1-stimulated group; n=3.