Fig 2: RelA-dependent JunB and ELK-1 signals exert transcriptional control of Nlrp3, Il1b, and Il18 expression.BMDMs were treated with LPS. (A) Immunoblotting analysis of the indicated proteins. (B) The expression of RelA mRNA in BMDMs was silenced by RNAi; after 48 hours, cells were further treated with LPS (1 µg/ml; 6 hours); qRT-PCR analysis of JunB and Elk1 gene expression. (C) WT and CD1d1-/- BMDMs were treated with LPS(1 µg/mL, 6h), iGb3 (5 µg/mL, 12h) or both, cell extracts were immunoblotted for the indicated molecules. (D) WT and CD1d1-/- BMDMs were treated with LPS (1 µg/ml; 6 hours), iGb3 (5 µg/ml; 12 hours) or both, and cell extracts were immunoblotted for the indicated molecules. (E) The JunB and (F) Elk1 genes in RAW264.7 cells were silenced by RNAi; after 48 hours, cells were further treated with LPS (1 µg/ml; 6 hours); immunoblotting analysis of the induced proteins and qRT-PCR detection of gene transcription. (G) ChIP-qPCR analysis for the enrichment of JunB and ELK-1 protein binding to the promoter regions of the indicated genes in WT BMDMs. Error bar, SEM; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, no significant difference (Student’s t test). One of three biological replicates, with three technical replicates.

Fig 3: H2S regulates TNFSF14 transcription through JNK/JunB signal.A Schematic overview of the predicted transcription factors of TNFSF14 gene by PROMO dataset (upper) and JunB binding sites on TNFSF14 promoter predicted with JASPAR website (lower). B ChIP assays for JunB binding onto TNFSF14 promoter were carried out in HepG2 (upper) and PLC/PRF/5 (lower) cells. The right panel showed the statistical analysis for the ChIP assay. C NaHS was used to treat HepG2 (left) and PLC/PRF/5 (right) cells for 0, 1, 2, 8, and 24 h. Expression of p-JunB in nuclear of HCC cells was detecting using immunofluorescence staining, and Nuclei and p-JunB were stained with DAPI (blue) and FITC (green), respectively. The image is representative of three independent experiments. D The effects of different time of NaHS treatment on the expression of p-JunB in the nuclear and cytoplasm were analyzed with Western blotting in the HepG2 cells and PLC/PRF/5 cells. E The expression levels of p-JunB protein were detected by Western blotting in the HCC cells under exogenous and endogenous H2S treatment. F Western blotting for the effects of PPG (CSE inhibitor) and SP600125 (p-JNK inhibitor) in the expressions of JNK/p-JNK, JunB/p-JunB, and TNFSF14 proteins in HepG2 and PLC/PRF/5 cells. All Bars, ± SD; n = 3; *P < 0.05 compared with control group in HepG2 cells, #P < 0.05 compared with control group in PLC/PRF/5 cells.

Fig 4: A working model of lutein-mediated cardioprotection in Ang II-induced cardiac remodeling by inhibiting AP-1/IL-11 signaling.

Fig 5: Lutein suppressed Ang II-induced IL-11 expression by inhibiting the activity and expression of c-Jun/Jun-B in CFs. (A) Changes in the expression of genes belonging to the AP-1 family, as revealed by transcriptomic profiling data. (B) The mRNA expression levels of AP-1 family members, including Fosl1, c-Fos, c-Jun, Junb, and Jund, in CFs in response to different treatments (n = 4 in each group). (C) AP-1-luciferase activity was measured by an AP-1 transcription factor assay kit (n = 5 in each group). (D, E) Representative blots and quantitative analysis of IL-11 and ERK (n = 4 in each group). (F, G) Representative blots and quantitative analysis of p-c-Jun and p-Jun-B (n = 4 in each group). (H, I) Representative blots and quantitative analysis of c-Jun and Jun-B (n = 4 in each group). (J) Quantification of CF proliferation in response to different treatments as determined by CCK-8 assays (n = 5 in each group). (K) Representative images of the Transwell migration assay and quantification of migrated CFs of the indicated groups (n = 5 in each group). (L, M) Representative images and quantitative analysis showing the levels of superoxide anions as measured by DHE staining (n = 5 in each group). (N) Representative blots and (O) quantitative analysis of COL-1, TGF-ß1, NOX2 and NOX4 (n = 4 per group). *P < 0.05, **P < 0.01 and ***P < 0.001 compared to the Ang II group; #P < 0.05, ##P < 0.01 and ###P < 0.001 compared to the Ang II + Lu + Vector group.