Fig 1: HCC patients had high SCUBE1 expression and a poor prognosis.(A) IHC staining for SCUBE1 was performed in HCC tissues and paracancerous tissues. Scale bar, 200µm. (B) HCC sections were stained with a-SMA (Red), vimentin (Green) and SCUBE1 (Cyan) by mIHC. Scale bar, 100µm. (C) The SCUBE1 content in the serum of HCC patients and healthy people was detected. (D) Relative SCUBE1 mRNA expression was evaluated by RT–qPCR. (E) Survival analysis was performed using KM plotter (P = 0.046 using log-rank test). (F) SCUBE1 expression was detected in HCC tissues and paracancerous tissues. ß-Actin was used as an internal control (*P < 0.05, t test). Data are represented as mean ± SD of three independent experiments
Fig 2: SCUBE1 promotes tumorigenesis in vivo through the Shh pathway.(A) Differences in tumour sizes in nude mice from different subcutaneous implantation groups. (B) Growth curve of mouse subcutaneous tumours derived from different treated PLC cells at the indicated time points (**P < 0.01, ***P < 0.001 vs. PLC + CAFs2, one-way ANOVA). (C) Differences in tumour weights in nude mice from different subcutaneous implantation groups. (D) Sections of HCC tissues were visualized using HE staining. Scale bar, 200µm. (E) Ki-67 immunohistochemical staining in different groups. Immunohistochemical staining of Ki67 expression was quantified (**P < 0.01, ***P < 0.001 vs. PLC + CAFs2, one-way ANOVA). Scale bar, 200µm. (F) Western blot analysis of SCUBE1, Shh, Smo and Gli1 expression in different groups was performed. GAPDH was used as an internal control. (G) The mRNA expression levels of SCUBE1, Shh, Smo and Gli1 in different groups were measured. ß-Actin was used as an internal control (**P < 0.01, ***P < 0.001 vs. CAFs2, one-way ANOVA). Data are represented as mean ± SD of three independent experiments
Fig 3: CAFs-derived SCUBE1 can regulate the malignant progression of HCC cells.(A) PLC and Huh7 cells were cocultured with CAFs1, CAFs2 and CAFs3 with or without anti-SCUBE1 antibody. Cell viability was measured with CCK-8 assays (***P < 0.001 vs. CAFs2 or CAFs3, one-way ANOVA). (B) RT–qPCR was performed to determine the mRNA levels of CD133, Sox2 and Nanog in PLC and Huh7 cells cocultured with CAFs1 and CAFs2 (*P < 0.05, t test). (C) The expression levels of CD133, Sox2 and Nanog in treated and untreated HCC cells were evaluated by Western blotting (***P < 0.001, t test). (D) CD133 was detected via immunofluorescence staining in different treated HCC cells (**P < 0.01, t test). Scale bar, 10µm. (E) The effects of CAFs1, CAFs2 and anti-SCUBE1 antibody on the colony formation ability of PLC and Huh7 cells (*P < 0.05, **P < 0.01, ***P < 0.001 vs. CAFs2, one-way ANOVA). (F) HCC cell migration ability was evaluated with Transwell assays after coculture with CAFs1 and CAFs2 with or without an anti-SCUBE1 antibody (**P < 0.01, ***P < 0.001 vs. CAFs2, one-way ANOVA). Scale bar, 200µm. Data are represented as mean ± SD of three independent experiments
Fig 4: SCUBE1 was highly expressed in CAFs and associated with stemness.(A) Gene changes in LX2 cells and cocultured LX2 cells were detected by transcriptome sequencing. (B) KEGG pathway enrichment analysis and GO functional annotation were used. (C) a-SMA, vimentin and CD31 expression in CAFs and NFs was detected by immunofluorescence staining (**P < 0.01, ***P < 0.001, t test). Scale bar, 10µm. (D) Western blotting was used to detect the mRNA expression of SCUBE1 in different CAFs and NFs. (E) RT–qPCR was used to detect the mRNA expression of SCUBE1 in different CAFs and corresponding NFs (**P < 0.01, ***P < 0.001, t test). (F) The secretion of SCUBE1 in the supernatants of different NFs and CAFs was detected by ELISAs (***P < 0.001, t test). (G) Kaplan–Meier analysis was performed to compare the low SCUBE1 expression group and the high SCUBE1 expression group. Data are represented as mean ± SD of three independent experiments
Fig 5: Effects of altered SCUBE1 expression in CAFs on the malignancy of HCC cells.(A) WB was used to detect the transfection efficiency of the overexpression plasmid and knockout plasmid. (B) RT–qPCR was used to detect the transfection efficiency of the overexpression plasmid (***P < 0.001 t test) and knockout plasmid (*P < 0.05, **P < 0.01, ***P < 0.001 vs. sh-Control, one-way ANOVA). (C) Cell viability was measured with CCK-8 assays in HCC cells subjected to different treatments (***P < 0.001, t test). (D) The effect of SCUBE1 silencing and overexpression in CAFs on the migration of HCC cells was measured with Transwell assays (**P < 0.01, t test). Scale bar, 200µm. (E) Sphere formation ability was detected in PLC cells and Huh7 cells. Scale bar, 500µm. (F) Shh, Smo, and Gli1 protein expression levels were detected via Western blotting in PLC and Huh7 cells cocultured with different treated CAFs. (G) Growth curve of mouse subcutaneous tumours derived from different treated PLC cells at the indicated time points (***P < 0.001, t test). Data are represented as mean ± SD of three independent experiments
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