Fig 2: TGF-ß1 induces transcriptional activation of Siglec-15 via MAPK signaling pathway. (A) Enrichment analysis of signaling pathways associated with Siglec-15 using The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) dataset through GTBA platform. (B,C) The signaling pathway enrichment analysis (B) and correlation analysis (C) between Siglec-15 and TGF-ß based on GTBA analysis using CCLE liver data source. (D) The expression levels of Ras/C-Raf/MEK/ERK1/2 signaling molecules, ETS-1/ETS-2 and their phosphorylation as well as Siglec-15 in HepG2 cells by Western blot analysis. (E) Effect of U0126-EtOH, a highly selective MEK1/2 inhibitor, on the expression of ERK1/2, ETS-1/2 and their phosphorylated forms as well as well Siglec-15 in HepG2 cells by Western blot analysis. (F) Schematic model for the mechanism by which TGF-ß1 activates Ras/C-Raf/MEK/ERK1/2 signaling to phosphorylate and activate ETS-1 and ETS-2 leading to upregulation of Siglec-15 expression. * p < 0.05. NC stands for untreated control.

Fig 3: Putative transcription factors and binding sites in the Siglec-15 promoter. (A) Activity analysis of the Siglec-15 promoter. HepG2 cells were transfected with each of the Siglec-15 promoter constructs and pRL-TK. The relative luciferase units (RLU) were obtained by comparison with the full-length Siglec-15 promoter construct (pGL4.10-1800-Luc). Each transfection was performed in duplicate and the data are expressed as mean ± SD of three independent experiments. (B) Nucleotide sequence of the -453 to -232 region of the Siglec-15 promoter. The six putative binding sites of ETS-1 and ETS-2 were underlined in the sequence and schematically depicted below. (C,D) Western blot analysis of ETS-1 and ETS-2 expression in HepG2 and Huh7 cells with ETS-1 or ETS-2 overexpressed (C) or knocked down (D). (E,F) Luciferase activities in the cells with ETS-1 or ETS-2 overexpressed (E) or knocked down (F). (G) Effect of EBS mutations on Siglec-15 pGL4.10-453 promoter activity. EBS mutants (0.25 µg) together with 10 ng of pRL-TK were co-transfected into HepG2 cells. The RLU were obtained by comparison with the wild-type pGL4.10-453-Luc. (H) Electrophoretic mobility shift assays with specific antibodies against ETS-1 and ETS-2. Arrowheads indicate the shift band (SB). The biotin-labeled probe was incubated in the absence (Lane 1) or presence (Lane 2) of nuclear extracts from HepG2 cells; unlabeled cold probe (Lane 3) and mutated probe (Lane 4) were used as competitor at a concentration of 100-fold molar excess to biotin-labeled probe. Super-shift assays were carried out with 8 µg specific antibody raised against ETS-1 or ETS-2 (Lane 5 or Lane 6). * p < 0.05; ** p < 0.01. NC stands for negative control.

Fig 4: TGF-ß1 upregulated the expression of Siglec-15 in HepG2 cells by facilitating the binding of ETS-1 and ETS-2 to EBSs in the Siglec-15 promoter. (A) Activity analysis of the Siglec-15 promoter in HepG2 cells treated with TGF-ß1 (50 ng/mL) for 24 h and then transfected with each of the Siglec-15 promoter constructs and pRL-TK. (B) Luciferase activity in HepG2 cells treated with TGF-ß1 (50 ng/mL) for 24 h and then transfected with pGL4.10-231-Luc, pGL4.10-453-Luc or the mutant plasmid with all the six EBSs mutated. (C) Nucleotide sequence of the -231 to -22 nt region of the Siglec-15 promoter. The five putative transcription factor binding sites are underlined in the sequence and schematically depicted below. (D,E) Effect of TGF-ß1 on Siglec-15 promoter activity in HepG2 cells with ETS-1 or ETS-2 overexpression or knockdown. pcDNA3.1-ETS-1, pcDNA3.1-ETS-2, si-ETS-1, or si-ETS-2 was co-transfected with pGL4.10-231-Luc into HepG2 cells treated with TGF-ß1 (50 ng/mL) for 24 h, then luciferase activities were measured 48 h after transfection. The RLU were obtained by comparison with empty vector pcDNA3.1/Hygro (+) or siRNA negative control (NC), respectively. (F,G) Effect of EBS mutation or deletion on TGF-ß1-induced Siglec-15 promoter activity. The variants of EBS mutations or deletions (0.25 µg) were co-transfected with 10 ng of pRL-TK into HepG2 cells treated with TGF-ß1 (50 ng/mL) for 24 h. The RLU was calculated by normalizing with the wild-type pGL4.10-231-Luc. (H) Electrophoretic mobility shift assays with specific antibodies against ETS-1 and ETS-2. Arrowheads indicate the shift band (SB). The biotin-labeled probe was incubated in the absence (Lane 1) or presence (Lane 2) of nuclear extracts from HepG2 cells treated with TGF-ß1 (50 ng/mL) for 24 h. Unlabeled cold probe (Lane 3) or mutated probe (Lane 4) was used as a competitor at a concentration of 100-fold molar excess to biotin-labeled probe. Super-shift assays were carried out with 8 µg specific antibody raised against ETS-1 or ETS-2 (Lane 5 or Lane 6). (I,J) Effect of ETS-1, ETS-2 and TGF-ß1 on Siglec-15 mRNA and protein expression in HepG2 cells determined by quantitative RT-PCR (I) and Western blot analysis (J). * p < 0.05; ** p < 0.01. ns stands for not significant and NC for untreated control.

Fig 5: ETS-1 and ETS-2 enhances transcription and protein expression of Siglec-15 in HepG2 and Huh7 cells. (A,B) Effect of ETS-1 or ETS-2 overexpression (A) or knockdown (B) on Siglec-15 expression in the transfected HepG2 or Huh7 cells by quantitative real-time PCR. (C,D) Effect of ETS-1 or ETS-2 overexpression (C) or knockdown (D) on Siglec-15 expression in the transfected HepG2 or Huh7 cells by Western blot analysis. * p < 0.05; ** p < 0.01. NC stands for negative control.