Figures & data
Figure 1 BHLHE41 overexpression enhances U87 and U251 cell proliferation and colony formation.
Notes: (A) BHLHE41 plasmid validity was confirmed using WB analysis, GAPDH served as the internal control; (B and C) CCK8 assays and Soft agar colony-formation assays were performed to analyze the BHLHE41 role in cell proliferation and colony formation. Compared with the control group, BHLHE41 overexpression significantly enhanced U87 and U251 cell proliferation and colony formation; (D and E) Cell cycle analysis of U87 and U251 cell lines with BHLHE41 overexpression. The x-axis represents the cell cycle distribution, and the y-axis represents the cell number. Cell percentages in G1/G0 phase was decreased, while S and G2 phase percentages increased after transfected with BHLHE41. A representative image of at least three independent experiments with similar results is shown. Each value represents the mean ± SD (bars) of three independent experiments; *P<0.05, **P<0.01, ***P<0.001.
Abbreviations: BHLHE41, Basic helix-loop-helix family member e41; WB, Western blot.
Figure 2 BHLHE41 silence lessens U87 and U251 cell proliferation and colony formation.
Notes: (A) Three kinds of siRNA were prepared for use with BHLHE41. Their efficiencies were confirmed by WB analysis. BHLHE41 siRNA#2 was selected as it had the greatest inhibitory effect. GAPDH served as the internal control; (B and C) Cell proliferation was assessed after BHLHE41 knockdown in U87 and U251 cell lines with CCK8 assays and Colony-formation assays; (D and E) Cell cycle analysis of U87 and U251 cell lines with BHLHE41 knockdown. The x-axis represents the cell cycle distribution, and the y-axis represents the cell number. A representative image of at least three independent experiments with similar results is shown. Each value represents the mean ± SD (bars) of three independent experiments; *P<0.05, **P<0.01, ***P<0.001.
Abbreviations: BHLHE41, Basic helix-loop-helix family member e41; WB, Western blot; NC, scrambled siRNA; siBHLHE41, BHLHE41 siRNA#2.
Figure 3 BHLHE41 regulates cyclin expression.
Notes: (A and B) WB analysis was performed to assess cyclin expression in U87 and U251 cell lines after BHLHE41 overexpression. CyclinD1 and cyclinE1 significantly upregulated after BHLHE41 transfection in U251 cells. CyclinD1 and cyclinD3 significantly upregulated in U87 cells, GAPDH served as the internal control; (C and D) WB analysis was performed to assess cyclin expression in U87 and U251 cell lines after silencing endogenous BHLHE41, GAPDH served as the internal control. (A representative image of at least three independent experiments with similar results is shown. Each value represents the mean ± SD (bars) of three independent experiments; *P<0.05, **P<0.01. Abbreviations: BHLHE41, Basic helix-loop-helix family member e41; WB, Western blot.
Figure 4 BHLHE41 promotes cell proliferation through the ERK/cyclinD1 pathway.
Notes: (A–F) A set of core signaling pathway-related proteins were measured by WB analysis after BHLHE41 overexpression (A, B and C) or knockdown (D, E and F) in U87 and U251 cell lines; (G-I) U87 and U251 cells were treated with 2000 nM SCH772984 for 6 h, then ERK, p-ERK, BHLHE41 and cyclinD1 were tested using WB analysis. GAPDH served as the internal control. SCH772984 significantly reduces p-ERK expression. The BHLHE41-induced cyclinD1 upregulation was reversed after blocking ERK pathway in both U87 and U251 cells. A representative image of at least three independent experiments with similar results is shown. Each value represents the mean ± SD (bars) of three independent experiments; *P<0.05, **P<0.01.
Abbreviations: BHLHE41, Basic helix-loop-helix family member e41; WB, Western blot.
Figure 5 BHLHE41 overexpression has no effect on cell apoptosis in U87 and U251 cells.
Notes: (A, B) U87 and U251 cells were transfected for 24 h with BHLHE41 or pcDNA, then transfected cells were treated with DMSO or cisplatin (50, 100, 150 μg/ml) for an additional 20 h. The number of apoptosis cells were measured by Annexin V-FITC/PI double staining assay. Data are presented as the mean ± standard deviation, from three independent experiments.
Abbreviations: BHLHE41, Basic helix-loop-helix family member e41; DMSO, Dimethyl sulfoxide; PI, propidium iodide.
Figure 6 BHLHE41 overexpression has no effect on cell apoptosis in U87 and U251 cells. (A, B) WB analysis was performed to assess apoptosis-related protein expression levels of Bax and Bcl-2 expression in BHLHE41 overexpression plasmid or pcDNA transfected U87 and U251 cells after treated with DMSO or cisplatin (50, 100, 150 μg/ml). GAPDH served as the internal control. Data are presented as the mean ± standard deviation, from three independent experiments.
Abbreviations: BHLHE41, Basic helix-loop-helix family member e41; WB, Western blot.
Table 1 Correlation of BHLHE41 expression with clinicopathological features in 509 LGG cases
Table 2 Correlation of BHLHE41 expression with clinicopathological features in 153 GBM cases
Figure 7 Correlation between BHLHE41 expression and the OS of 509 LGG cases and 153 GBM cases.
Notes: (A) The OS of LGG patients with high level of BHLHE41 was much lower than that of group with low to median level of BHLHE41(P=0.0019); (B) The OS of GBM patients with high level of BHLHE41 was lower than that of the patients with low to median level of BHLHE41, but without statistical difference (P=0.17); (C) High level of BHLHE41 indicates poor prognosis of the patients with WHO grades 2 and 3 (P<0.0001).
Abbreviations: BHLHE41, Basic helix-loop-helix family member e41; LGG, low-grade glioma; GBM, glioblastoma; OS, overall survival.
Figure S1 Expression of BHLHE41 in different organs.
Notes: Reproduced from Human Protein Atlas. Available from: https://www.proteinatlas.org/ENSG00000123095-BHLHE41/tissue. Creative Commons license and disclaimer available from: http://creativecommons.org/licenses/by/4.0/legalcode.Citation1 The data demonstrated that BHLHE41 had the highest expression in U138, U251 and U87 cell lines in brain.
Abbreviation: BHLHE41, Basic helix-loop-helix family member e41.
