Figure 8.

GSCAR-targeting ASO impeded tumor growth. (A-B) GSCAR knockdown promoted TMZ-induced cellular apoptosis in GSC11 cells as detected by flow cytometry (A) and SRB (B) assays. Quantification results are indicated. (C) Marker genes related to cellular apoptosis were detected by immunoblotting with the indicated antibodies. (D) The relative expression of GSCAR was examined by RT -PCR after transfection with the indicated ASO. (E-F) GSCAR-targeting ASO inhibited cell proliferation, as examined by growth curve (E) and colony formation (F) assays. Quantification results are indicated. (G) GSCAR-targeting ASO repressed cell migration and invasion in the transwell assay. Scale bar=50 μm. (H) GSCAR-targeting ASO inhibited cancer stem cell self-renewal ability by tumorsphere formation assay. Quantification results are indicated. Scale bar=50 μm. (I) GSCAR wild-type but not mutant was able to rescue GSCAR-targeting ASO reduced cell growth phenotype. GSCAR-WT=GSCAR wild-type; GSCAR-MUT=GSCAR mutant=GSCAR targeting ASO insensitive mutant. (J) Schematic view of the xenograft mouse model treated with the indicated ASO (5 nM) and TMZ (60 mg/kg). (K-M) Representative xenograft tumor images (K), tumor masses (L), and tumor volumes (M) are shown for the indicated groups treated with the indicated ASO with or without TMZ. GSC11 cells were used. (N) Quantified results for the IHC staining of Ki67 and CC3 are presented in the indicated xenograft tumor sections. (O) Schematic diagram showing how GSCAR promotes glioma progression via both the GSCAR/miR-6760-5p/SRSF1 and GSCAR/DHX9-IGF2BP2/SOX2 axes. * P < 0.05, ** P < 0.01, *** P < 0.001. NC-ASO = ASO negative control; GR-ASO=GSCAR targeting ASO.