Article Figures & Data
Figures
- Figure 1
The changes in BYSL expression in glioma tissues and association of BYSL levels with patient survival. (A) Expression analysis of the GSE16011 dataset revealed a significant increase in the BYSL mRNA levels in grade II, grade III, and grade IV glioma tissues compared to those in nontumor tissues. (B, C) Expression analysis with the GEPIA showed that BYSL expression was significantly higher in low grade gliomas and in glioblastomas than those in their respective controls. T: tumor; N: normal. (D) The qRT-PCR analysis showed the upregulation of the BYSL mRNA levels in grade II, grade III, and grade IV glioma tissues compared to those in nontumor tissues. (E, F) Western blot analysis showed an increase in BYSL protein levels in grade II, grade III, and grade IV glioma tissues compared to those in nontumor tissues. (G, H) Immunohistochemistry showed that BYSL immunoreactive (BYSL-IR) signals were distributed in both the cytoplasm and nucleus of tumor cells (G). Scale bars: 100 μm for a and c; 20 μm for b and d. Quantitative analysis of the cell numbers showed increased percentages of BYSL-IR cells in grade II (n = 4), grade III (n = 4), and grade IV (n = 5) glioma tissues relative to those in nontumor tissues (n = 5) (H). (I, J) Survival analysis showed that high BYSL levels were associated with poor prognoses in both TCGA (I) and CGGA (J) glioma datasets. M, molecular marker (*P < 0.05; **P < 0.01; ***P < 0.001).
- Figure 2
The efficiency of BYSL downregulation and overexpression in glioma cells. (A) The qRT-PCR revealed that different concentrations of BYSL siRNAs significantly downregulated the BYSL levels in U251 and U87 glioma cells. (B) Western blot analysis showed that 100 nM siRNA significantly decreased the BYSL protein levels in U251 and U87 glioma cells. (C) By combining the green fluorescent protein and 4,6-diamidino- 2-phenylindole staining, 90% of the cells were confirmed to be infected by lentiviruses produced from the vector and BYSL plasmids. Scale bar: 200 μm. (D) Western blot analysis showed that BYSL was abundantly expressed in U251 and U87 cells. M, molecular marker (*P < 0.05; **P < 0.01; ***P < 0.001).
- Figure 3
The effects of BYSL downregulation or overexpression on glioma cell growth. (A, B) CCK-8 assays were used to measure the cell viability at 24, 48, and 72 h following BYSL downregulation (A) or overexpression (B) in U251 and U87 cells. (C–E) The 5-ethynyl-20-deoxyuridine (EdU) assays were performed to evaluate the cell proliferation in U251 and U87 cells. Representative images of BYSL downregulation and overexpression are shown in C and D, respectively. Scale bars: 200 μm. Quantitative analyses of the EdU-positive cells with BYSL downregulation and overexpression are shown in E. (F) CCK-8 assays were used to measure the cell viability in primary glioma cells at 24, 48, and 72 h following BYSL downregulation (*P < 0.05; **P < 0.01; ***P < 0.001).
- Figure 4
The effects of BYSL downregulation or overexpression on cell cycle progression in glioma cells. (A–D) Flow cytometry was used to analyze the cell cycle progression in U251 and U87 cells. Representative histograms of BYSL downregulation and overexpression are shown in A and C, respectively. Quantitative analyses of the percentage of cells in G1, S, and G2 phases are shown in B (BYSL downregulation) and D (BYSL overexpression). (E–H) Western blot analysis was used to measure the changes in the cell cycle-related proteins in U251 and U87 cells. Representative blot images of BYSL downregulation and overexpression are shown in E and G, respectively. Graphs quantifying the blot images of BYSL downregulation and overexpression are shown in F and H, respectively. M, molecular marker (*P < 0.05; **P < 0.01; ***P < 0.001).
- Figure 5
The effects of BYSL downregulation on glioma cell apoptosis. (A, B) Flow cytometry was used to analyze apoptosis in U251 and U87 cells. Representative histograms are shown in A. Quantitative analyses of the percentage of apoptotic cells are shown in B. (C, D) Western blot analyses of caspase-3 and PARP levels were used to evaluate apoptosis in U251 and U87 cells. Representative blot images are shown in C. Graphs quantifying the blot images are shown in D. M, molecular marker (*P < 0.05; **P < 0.01).
- Figure 6
The association of BYSL with RIOK2 and mTOR. (A) FLAG-BYSL plasmid was transfected into HEK293T cells. Immunoprecipitation (IP) with FLAG magnetic beads showed that mTOR and RIOK2 were associated with BYSL. (B) The Myc-RIOK2 plasmid was transfected into HEK293T cells. IP with the anti-Myc antibody showed that mTOR and BYSL were associated with RIOK2. (C) FLAG-BYSL plasmid was transfected into HEK293T cells. IP with the anti-mTOR antibody showed that RIOK2 and BYSL were associated with mTOR. (D) IP with the anti-BYSL antibody showed the association of BYSL with RIOK2 and mTOR in U251 cells. (E) Double labeling immunofluorescence was performed in U251 glioma cells. BYSL partially co-localized with RIOK2 in both the cytoplasm and nuclei. Scale bar: 20 μm. (F, G) Correlation analysis between BYSL and RIOK2 at the gene level in the LGG, GBM, and GSE16011 datasets. (H) Correlation analysis between BYSL and RIOK2 at the protein level in our glioma samples. The correlation efficiency and P-values are indicated in the figures. M, molecular marker.
- Figure 7
The effects of BYSL overexpression or downregulation on the AKT and mTOR signaling pathways in glioma cells. Western blot analysis was used to measure the levels of the total and phosphorylated AKT/mTOR signaling molecules in U251 and U87 cells. Representative blot images of BYSL overexpression and downregulation are shown in A and C, respectively. Graphs quantifying the blot images of BYSL overexpression and downregulation are shown in B and D, respectively. M, molecular marker (*P < 0.05; **P < 0.01; ***P < 0.001).
- Figure 8
The effects of BYSL or RIOK2 downregulation on glioma growth in vivo. An orthotopic xenograft model was established in nude mice. (A–D) Hematoxylin and eosin (H&E) staining was performed with whole brain sections from tumor-bearing mice. Representative H&E-stained images for the shRIOK2-2 and shBYSL groups are shown in A and C, respectively (100×). Graphs showed that silencing RIOK2 (B) or BYSL (D) significantly decreased the tumor volume. (E, F) Survival analysis showed that silencing RIOK2 or BYSL significantly prolonged the overall survival time of tumor-bearing mice. (G) A working model. BYSL and RIOK2 exist in the same complex with mTORC2, which is in turn connected to the AKT-mTORC1-P70S6K signaling, and thus increases ribosomal protein (e.g., S6) synthesis, leading to glioma cell growth and proliferation (*P < 0.05; **P < 0.01).
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