Fig 1: Morphology and GPC3 expression of primary and metastatic osteosarcoma tissues and their spheroids. (A) H&E staining of primary and metastatic OS tissue under different magnification. (B) Bright field and HE demonstration of primary and metastatic OS tissues and their spheroids (X 20). (C) SOX9 (green) and vimentin (red); (D) GPC3 (green) and CD133 (red) immunofluorescence imaging of the primary and metastatic tumors and their spheroids (X10). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig 2: Schematic diagram of osteosarcoma patient’s evolution tree. GPC3 mutation abundance was 56.49% in the primary tumor and 87.06% in the metastatic one, indicating that the mutant GPC3 harboring OS cells became predominant as tumor progression.
Fig 3: GPC3 targeted therapy performed on metastatic OS spheroids. (A) Apoptosis and proliferation of m-OS spheroids labeled by TUNEL and EdU under anti-GPC3-antibody (GPC3-Ab) treatment. (B) Cell death rate of m-OS spheroids treated by gradient GPC3-Ab, 4 µM cisplatin. and GPC3-Ab/cisplatin. combination. *, p = 0.013 in comparison with normal culture group; **, p = 0.00046, 0.00049, 0.00026, in comparison with normal culture group; #, p = 8.44x10-5 in comparison with in comparison with 2.0 µg/mL GPC-Ab treated group.
Fig 4: Screening of target genes regulated by miRNA-96-5p and the effect of up-regulation of GPC3 expression on radiation reactivity of HR-8348 cells. (A–C) qRT-PCR assays were used to detect the expression levels of target genes in rectal cancer cells before and after miRNA-96-5p knockdown, and the mRNA expressions of FOXO3 and GPC3 were significantly up-regulated in HR-8348-IN cells; (D–G) Western blot assays were used to detect the expression of target genes in rectal cancer cells before and after miRNA-96-5p knockdown, and the protein expression of GPC3 was not significantly changed in HR-8348-NC cells, but was significantly up-regulated in HR-8348-IN cells; (H) Luciferase activity of the WT and mutant GPC3 3'UTR co-transfected with miRNA-NC and miRNA-96-5p mimics, the GPC3-WT group was inhibited, but not in the GPC3-MUT group; (I) GPC3 was significantly up-regulated in HR-8348-GPC3 cells; (J) GPC3 over-expression in HR-8348 cells results in increased sensitivity to irradiation, and survival fraction fitted to the linear quadratic equation. **P < 0.01.
Fig 5: Increased abundance of GPC3 mutation in the metastatic osteosarcoma. (A) Reverse transcription polymerase chain reaction detected GPC3 expression and Gray value analysis of GPC3 expression between the primary and metastatic OS tissue and their spheroids. (B) qPCR quantification of GPC3 expression in primary and metastatic OS tissues and spheroids. (C) Sanger sequencing analysis of GPC3 transcripts demonstrated G to A mutation in Exon 4.
Supplier Page from Abcam for Anti-Glypican 3 antibody [EPR10641]