Fig 2: FXR promotes Jak2/STAT3-mediated NSCLC metastasis and angiogenesis by upregulating IL-6 and IL-6ST.ELISA showing the concentrations of IL-6 in the culture supernatant of FXR-silenced A549 and H1975 cells (A) and FXR-overexpressed A549 stable cells treated with increasing concentrations of Z-GS (0, 10, 20, and 40 µM) (B). Western blotting was performed to evaluate the protein levels of IL-6 and IL-6ST in FXR-silenced A549 and H1975 cells (C) and FXR-overexpressed A549 stable cells treated with increasing concentrations of Z-GS (0, 10, 20, and 40 µM) (D). GAPDH was used as a loading control. qRT-PCR was performed to examine the mRNA levels of IL-6 and IL-6ST in FXR-silenced A549 and H1975 cells (E) and FXR-overexpressed A549 stable cells treated with increasing concentrations of Z-GS (0, 10, 20, and 40 µM) (F). β-actin served as an internal control. G–I FXR-overexpressed A549 stable cells were transfected with NC- or IL-6ST-siRNAs for 48 h. G Expression levels of IL-6ST, p-Jak2 (Tyr1007/1008), t-Jak2, p-STAT3 (Tyr705), and t-STAT3 protein were examined by Western blotting. H Cell migratory and invasive abilities were assessed by Transwell assays. I Cell angiogenic abilities were evaluated by angiogenesis assays. For (H, I), representative images (magnification, ×200) and quantitative results are shown. Data represents mean ± SD from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, compared with the NC or mock group. †p < 0.05, ††p < 0.01, †††p < 0.001, compared with A549-FXR group. NS, not significant.

Fig 3: CYLD interacted with and stabilized YAP protein by deubiquitination.A GSEA in DU145 sublines (DU145 shCon and DU145 shCYLD) detected by RNA-seq results showing that “WP_HIPPO_SIGNALING” responded to low CYLD expression. B Western blot analysis of YAP expression levels in these established cell lines. C Quantitative real-time PCR (qRT-PCR) analysis of the mRNA expression levels of YAP in these established cell lines (N = 3). D Co-immunoprecipitation (Co-IP) assay was applied to validate the relationship between CYLD and YAP proteins in 293 T and PC-3 cells. E 293 T cells transfected with YAP and Ubi were pretreated with MG132 (20 μM) for 6 h, the ubiquitination assay was performed with immunoprecipitation by YAP antibody and analyzed by Western blot. F DU145 sublines were pretreated with MG132 (20 μM) and Ubi for 6 h, the ubiquitination assay was performed with immunoprecipitation by YAP antibody and analyzed by Western blot. G, H DU145 and PC-3 sublines were treated with 100 μg/ml CHX for the indicated time. Equal amounts of WCL were immunoblotted with the indicated antibodies. GAPDH was used as the loading control. I, J The YAP protein abundance was quantified by ImageLab and plotted as indicated. GAPDH was used as the loading control.

Fig 4: α-Hederin activates incomplete autophagy in NSCLC by inhibiting mTOR. (A) α-Hederin(16 μM) treatment of H1299 and A549 for 24 h followed by immunofluorescence assay to label LC3 protein. Scale bar 20 μm. (B) α-Hederin(16 μM) treatment of H1299 and A549 for 24 h followed by Western blotting to detect LC3 and p62 protein expression, with GAPDH as an internal reference. (C) α-Hederin or Rapamycin(100 nM) treatment of H1299 and A549 for 24 h followed by Western blotting to detect mTOR protein expression, with GAPDH as an internal reference. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. Ctrl (Control).

Fig 5: CYLD suppressed PCa cell proliferation in vitro and in vivo.A Western blot analysis of CYLD expression levels in human PCa cell lines and the normal prostate cell line RWPE-1. GAPDH was used as the loading control. B Quantitative real-time PCR (qRT-PCR) analysis of the CYLD mRNA expression levels in PCa cell lines and the normal prostate cell line. 18 S was applied as the endogenous control. Data are presented as the mean ± SEM, n = 3. C Western blot analysis of CYLD expression in DU145 cell lines transfected with CYLD shRNAs and shControl. D Western blot analysis of CYLD expression in 22RV1 and PC-3 cell lines transfected with CYLD lentivirus and negative control. E Cell viability was detected by MTT assay in DU145 cell lines transfected with CYLD shRNAs and shControl. F Colony formation of DU145 cell lines transfected with CYLD shRNAs and shControl, and quantitative analysis was shown (N = 3). G Cell viability was detected by MTT assay in 22RV1 transfected with CYLD lentivirus and negative control. H Cell viability was detected by MTT assay in PC-3 transfected with CYLD lentivirus and negative control. I, J, K L Images of the tumor growth of subcutaneous xenografts established by CYLD overexpressing 22RV1 sublines. The wet weights of xenografts were measured after harvesting and tumor volumes were measured every three days. IHC analysis of the expression levels of CYLD and Ki67 in xenografts was shown. (**p < 0.01; ***p < 0.001).