Fig 1: CYLD is critical for protection against the receptor-interacting protein kinase 1 (RIPK1)-induced apoptosis in a subset of melanoma cells. (A) Mel-CV, ME1007, Mel-FH, and ME4405 cell lines transfected with indicated siRNAs were subjected to Western blotting. Data shown are representative of three individual experiments. (B) Immunoprecipitates with RIPK1 antibody from Mel-CV and ME1007 cells transfected with indicated siRNAs and plasmids were subjected to Western blotting. Data shown are representative of three individual experiments. (C) Whole-cell lysates from Mel-CV and ME1007 cell lines transfected with indicated plasmids with or without treatment with MG132 were subjected to Western blotting. Data shown are representative of three individual experiments. (D) Mel-CV and ME1007 cells transfected with indicated siRNAs were subjected to propidium iodide (PI)/annexin V staining assays. Data shown are representative of three individual experiments (left) or means ± SEM (right). n = 3. ***p < 0.001, Student’s t-test. (E) ME4405 and Mel-FH cells transfected with indicated siRNAs were subjected to CellTiter-Glo assays. Data shown are means ± SEM. *p < 0.05; **p < 0.01; ns, p > 0.05, Student’s t-test. (F) Whole-cell lysates from Mel-CV and Mel-FH cells were subjected to Western blotting. Data shown are representative of three individual experiments. (G) Mel-CV and ME1007 cells with CYLD knocked down by siRNA were transfected with the indicated plasmids were subjected to PI/annexin V staining assays (top) and Western blotting (bottom). Data shown are means ±SEM (top) or representative of three individual experiments (bottom). n = 3. **p < 0.01; ***p < 0.001, Student’s t-test.
Fig 2: Mechanistic map of blocking the Notch pathway to improve cardiac fibrosis. Blocking of the Notch signaling pathway promoted M2 macrophage polarization and increased CYLD expression in macrophages, followed by inhibiting the activation of NF-?B pathway, inflammatory factors, and fibrosis-related factors, and ultimately suppressed cardiac fibrosis remodeling.
Fig 3: Blocking the Notch signaling inhibits the activation of the NF-?B pathway. (A): p65 levels in mouse hearts were detected by immunohistochemistry staining; (B): Phosphorylation level of the NF-?B pathway was detected by Western blot; (C): mRNA expression of CYLD in mouse cardiac macrophages was detected by qRT-PCR; (D): Protein expression of CYLD in mouse cardiac macrophages was detected by Western blot; N = 6; three independently repeated tests were performed, and the data were expressed as mean ± SD. One-way ANOVA was used for the analysis of variance, and Tukey's multiple comparisons test was used for post hoc test; ** P < 0.01 (compared with the sham group), and ## P < 0.01 (compared with the sh-NC group).
Fig 4: (A) Relative expression levels of miR-197-3p were significantly elevated in the mimics group compared with in the control groups. (B) MTT assay of HBE cells transfected with miR-197-3p mimics. Proliferation was significantly higher in the mimics group. *P<0.05 vs. blank group. (C) Apoptotic analysis of HBE cells transfected with miR-197-3p mimics. Caspase activity was decreased in the mimics group. *P<0.05 vs. blank group. (D) Putative miR-197-3p binding sites in the 3'-UTR of CYLD mRNA. (E) Effects of miR-197-3p mimics and inhibitors on CYLD expression in HCC827 cells, as detected using western blotting, 48 h post-transfection. GAPDH was used as a loading control. The expression levels of CYLD were markedly lower in the mimics group and higher in the inhibitors group compared with in the control groups (blank or NC groups). (F) Potential target genes in the overlap of the two gene sets. (G) Dual-luciferase reporter assays using vectors encoding putative miR-197 target sites in the CYLD 3'-UTR for both wt and mut type. In the pGLO-mut-CYLD groups, no significant difference was observed between the relative luciferase activity of the cells co-transfected with the miR-197-3p mimics and cells co-transfected with NC. Conversely, the relative luciferase activity of the groups transfected with pGLO-wt-CYLD + miR-197-3p was markedly lower than in the pGLO-wt-CYLD + NC group. *P<0.05 vs. NC + wild-type group Normalized data were calculated as Renilla/firefly luciferase activity. CYLD, lysine 63 deubiquitinase; miR, microRNA; mut, mutant; NC, negative control; OD, optical density; UTR, untranslated region; wt, wild-type.
Fig 5: Blocking of the Notch signaling pathway in vitro promoted M2 macrophage polarization and CYLD expression and inhibited fibrosis-related factors expression and cellular inflammatory levels. (A): Bone marrow-derived macrophages in mice were identified by flow cytometry; (B): Heart-derived fibroblasts were identified by immunohistochemistry. Macrophages with different treatments were isolated and divided into Mø-Sham, Mø-MI, Mø-sh-NC, and Mø-sh-RBP-J groups. Macrophages and fibroblasts stimulated with LPS + INF-? for 24 h were co-cultured by Transwell to establish an in vitro inflammatory model; (C): Macrophage polarization was detected by flow cytometry; (D): p65 in macrophage nuclei of each group was detected by immunofluorescence; (E): Expression of fibrosis-related factors TGF-ß1, PDGF-B, COL1, and COL3 in fibroblasts was detected by qRT-PCR; (F): Expression of inflammatory factors TNF-a, IL-1ß, and IL-6 in macrophages was detected by ELISA; (G): The expression of CYLD in macrophages was detected by qRT-PCR. Three independently repeated tests were performed, and the data were expressed as mean ± SD. One-way ANOVA was used for analysis of variance, and Tukey's multiple comparisons test was used for post-hoc test; ** P < 0.01, * P < 0.05 (compared with Mø-Sham group), and ## P < 0.01, # P < 0.05 (compared with Mø-sh-NC group).
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