Fig 1: Blocking the Shh pathway can reverse the stimulatory effect of SCUBE1.(A) Coimmunoprecipitation of SCUBE1 and Shh in PLC and Huh7 cells. The IgG isotype was used as a negative control. (B) The invasion capacity of PLC and Huh7 cells treated with rhSCUBE1 and IgG was detected with Transwell assays (**P < 0.01, t test). Scale bar, 200µm. (C) The expression of CD133 in different groups was measured via Western blotting. (D) The Shh content in the supernatant in PLC and Huh7 cells with different treatments was measured with ELISA (*P < 0.05, t test). (E) The proliferation rates of PLC and Huh7 cells after the addition of rhSCUBE1 with or without Shh antibody were measured (***P < 0.001 vs. rhSCUBE1, one-way ANOVA). (F) Western blot analysis of Smo and Gli1 expression in PLC and Huh7 cells treated with rhSCUBE1 and cyclopamine. ß-Actin was used as an internal control. (G) The invasion capacity of PLC and Huh7 cells treated with rhSCUBE1 and cyclopamine was measured with Transwell assays (**P < 0.01, t test). Scale bar, 200µm. 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: 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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