Fig 2: Western blots of RNA-expressed genes (RAF1, CNBP, ISY1) to detect protein in embryos (Stage 36 HH) of the three genotypes of the Wingless-2.331 line show that wg-2/wg-2 mutant embryos lack RAF1 protein expression. Precision Plus Protein Dual Color standards (Bio-Rad; Hercules, CA, USA) are shown at the left to indicate the approximate size of the bands. Dotted lines indicate cuts made in the PVDF membrane in order to incubate pieces separately using different primary antibodies. In total, n = 12 individuals (four each of: wild-type, carrier, and mutant) were examined for RAF1, ISY1, and CNBP protein expression as compared to GAPDH. Three technical replicates for each individual were performed. (a) shows a representative image of two biological replicates of each genotype with bands for RAF1 (above the dotted line, 73 kDa) and GAPDH (below the dotted line, 35 kDa). The average ratio of RAF1 expression compared to GAPDH in wild-type in this blot was 2.08, whereas this ratio was 0.91 in carriers and zero in mutants (calculated using ImageJ software, Version 1.51). This supports the visual assessment that carriers have roughly half the RAF1 expression of wild-type individuals. (b) shows a representative image of a wild-type, carrier, and two mutant individuals with bands for ISY1 (left blot, 33 kDa) and GAPDH (right blot, 35 kDa). Expression appears equivalent across samples, and ImageJ for this blot was used to calculate an ISY1/GAPDH ratio of 0.95 for wild-type, 1.13 for carrier, and 1.00 for the average of the two mutants. (c) shows a representative image of three wild-type, three carrier, and two mutant individuals for CNBP (bottom, 19 kDa) and GAPDH (top, 35 kDa). A strong band did not appear for CNBP even at high concentrations of primary antibody, and the faint band at the predicted weight (19 kDa) did not register above the background in most lanes, as analyzed by ImageJ.

Fig 3: Pro enhances the inhibitory binding of 14–3-3ζ to C-Raf and blocks the positive feedback loop of TGF-β1 in OB-educated PCa. A Immunoprecipitation of 14–3-3ζ by C-Raf in C4-2B cells treated with DMSO or Pro (400 nM). IgG was used as the negative control. B Representative IF images of subcellular location of 14–3-3ζ and C-Raf in C4-2B cells treated with DMSO or Pro (400 nM). Green and red arrows indicate the separate locations of 14–3-3ζ and C-Raf; yellow arrows indicate the co-localization of 14–3-3ζ and C-Raf. C Western blot of the phosphorylation level of proteins in C-Raf/MAPK pathway in C4-2B cells treated with DMSO or Pro (400 nM). D Western blot of the phosphorylation level of indicated proteins in co-cultured C4-2B cells treated with DMSO, SB-431542 (10 μM) or Pro (400 nM). E Western blot of TGF-β1 in co-cultured C4-2B cells treated with DMSO, PD98059 (10 μM) or Pro (400 nM). F Fold change of TGF-β1 mRNA in C4-2B cells cultured alone and co-cultured with MC3T3 and treated with TGF-β1 (10 ng/ml) and Pro (400 nM). G Western blot of the phosphorylation level of proteins in C-Raf/MAPK pathway with or without 14–3-3ζ knockdown under indicated conditions. H Western blot of TGF-β1 in C4-2B-sh140 cell treated with DMSO, PD98059 (10 μM) or Pro (400 nM). I IHC staining of 14–3-3ζ and TGF-β1 in serial bone sections. J Schematic of Pro binding to 14–3-3ζ and blocking the positive feedback loop of TGF-β. ***P < 0.001

Fig 4: Effects of hub gene expression on prognosis of patients with hepatocellular cancer. Expression of the top 5 hub genes was assessed: CDK1, CCND1, RAF1, CDKN1B and BTRC. A Overall survival (OS) of patients showing high and low expression of CDK1. B Disease-free survival (DFS)of patients showing high and low expression of CDK1. C Expression of CDK1 in patients with hepatocellular cancer and healthy volunteers, *p < 0.001. D Expression of CDK1 in patients with hepatocellular cancer at different stages. E OS and DFS in patients showing high or low expression of CCND1. F OS and DFS in patients showing high or low expression of RAF1. G OS and DFS in patients showing high or low expression of CDKN1B. H OS and DFS in patients showing high or low expression of BTRC

Fig 5: Arecoline treatment changes the expression of miRNAs and inflammatory factors via PI3K/AKT/mTOR pathwayin HepG2 hepatoma cells. All analyses were carried out with culture medium of HepG2 hepatoma cells treated with 2.5 μM arecoline and con group. A Exosomal miR-1267 expression. B Exosomal miR-21-3p expression. C Exosomal miR-21-5p expression. D IL-1β protein levels. E TNF-α protein levels. F Relative expression of mRNAs encoding PI3K/AKT/mTOR signaling proteins.G Western blot analysis of PI3K/AKT/mTOR signaling proteins. H Relative expression of PI3K/AKT/mTOR signaling proteins. I Western blot analysis of hub genes. J Relative expression of CDK1, CCND1, RAF1, CDKN1B and BTRC mRNA and protein. Vs CON group, ap < 0.05; vs 2.5 μM group, bp < 0.05; 5 μM group, cp < 0.05