Figure 1.
High G9a expression is a prognostic indicator of poor survival in neuroblastoma patients.
A, Kaplan–Meier analysis of progression-free survival for the Versteeg database with the log rank test P value indicated. B, box plot of G9a expression levels in stage (ST) 1–4S tumors. C, box plot of G9a expression levels in tumors from patients in the non-death and tumor-caused-death groups. D, box plot of G9a expression levels in tumors from patients in the <18-month and >18-month groups. E, Kaplan–Meier analysis of progression-free survival for the Neuroblastoma Prognosis Database with the log rank test P value indicated. F, box plot of G9a expression levels in tumors from dead and alive groups. G, western blot analysis of G9a expression in five neuroblastoma cell lines. For the data in A, the G9a cutoff value of 7.62 was used to separate the patients into high and low G9a expression groups. The data in B (ST1 vs. ST2, ST2 vs. ST3, ST3 vs. ST4, and ST4 vs. ST4S), C, D, and F were analyzed using two-tailed student's t-test with the P values indicated. For the data in E, the G9a cutoff value of 0 was used to separate the patients into high and low G9a expression groups.
Figure 2.
Inhibition of G9a represses neuroblastoma cell growth and proliferation.
A, morphologic examination of five neuroblastoma cells treated with BIX01294 or water for 2 and 4 days. Scale bars, 5 µm. B, neuroblastoma cells were either treated with 5 µM BIX01294 or water for 2 days and 4 days, respectively, then analyzed for cell counting with the TC10 Automated Cell Counter, error bars, SD, n = 5. Statistical analysis was performed using two-tailed student's t-test, *p≤0.01. C, neuroblastoma cells were either treated with 5 µM BIX01294 or water and then analyzed for cell growth by the CCK8 assay. Each value represents the average obtained from five independent experiments; error bars, SD. Statistical analysis was performed using two-tailed student's t-test, *p≤0.01.
Figure 3.
Inhibition of G9a induces cell cycle arrest in G1 phase.
A and B, neuroblastoma cells were either treated with 5 µM BIX01294 or water for 2 days and analyzed for the cell cycle by flow cytometry. Each column represents the average obtained from three independent experiments; error bars, SD. Statistical analysis was performed using two-tailed student's t-test, *p≤0.01. C, western blot analysis of cyclins and CDKs associated with G1 phase in neuroblastoma cells treated with BIX01294 or water for 2 days. α-Tubulin levels are shown as the loading control.
Figure 4.
Inhibition of G9a induces autophagy in neuroblastoma cells.
A, immunofluorescence analysis of five neuroblastoma cells treated with BIX01294 or water for 2 and 4 days. Scale bars, 5 µm. B, Statistical analysis of LC3B puncta in neuroblastoma cells treated with BIX01294 or water for 2 and 4 days. Each column represents the average obtained from three independent experiments; error bars, SD. Statistical analysis was performed using two-tailed student's t-test, *p≤0.01. C, western blot analysis of G9a function in neuroblastoma cells treated with BIX01294 or water for 2 days. D, western blot analysis of autophagy-related genes in neuroblastoma cells treated with BIX01294 or water for 2 days. α-Tubulin levels are shown as the loading control.
Figure 5.
Inhibition of G9a decreases the tumorigenicity of neuroblastoma cells.
A, neuroblastoma cells were plated at 1×103 to 2.5×103 cells per well in six-well culture plates. After 14 to 21 days of culture, soft agar colonies developed with cells treated with water. As shown, the cells treated with 5 µM BIX01294 were observed to give rise to small and scanty colonies in soft agar. Scale bars, 50 µm. B, colonies that were larger than 0.5 mm or that contained more than 50 cells were recorded. Each column represents the average obtained from three independent experiments; error bars, SD. Statistical analysis was performed using two-tailed student's t-test, *p≤0.01. C, tumor growth in NOD/SCID mice injected with the indicated neuroblastoma cells as measured by caliper measurements. D, scatter plot of xenograft tumor weight with horizontal lines indicating the mean per group. The data were analyzed with 2-tailed Student t test, and the P value is indicated.
Figure 6.
Downregulation of G9a represses neuroblastoma cell growth and proliferation.
A, western blot analysis of G9a in neuroblastoma cells BE(2)-C expressing GFPsi or individual G9asi sequences. α-Tubulin levels are shown as the loading control. B, morphological examination of three neuroblastoma cells expressing GFPsi, G9asi#3 or G9asi#4, respectively. The cells expressing GFPsi are shown as the biological controls. Scale bar, 5 µm. C, neuroblastoma cells expressing GFPsi, G9asi#3 or G9asi#4 were analyzed for cell counting using the TC10 Automated Cell Counter, error bars, SD, n = 5. Statistical analysis was performed using two-tailed student's t-test, *p≤0.01. D, proliferation assays of three neuroblastoma cells expressing GFPsi, G9asi#3 or G9asi#4, error bars, SD, n = 5. E, western blot analysis of cyclins and CDKs related to the G1 phase in neuroblastoma cells with G9a knockdown. α-Tubulin levels are shown as the loading control.
Figure 7.
Downregulation of G9a induces autophagy and decreases tumorigenicity in neuroblastoma cells.
A, immunofluorescence analysis of three neuroblastoma cells expressing GFPsi, G9asi#3 or G9asi#4. Scale bar, 5 µm. B, statistical analysis of LC3B puncta in neuroblastoma cells expressing GFPsi, G9asi#3 or G9asi#4. Each column represents the average obtained from three independent experiments; error bars, SD. Statistical analysis was performed using two-tailed student's t-test, *p≤0.01. C and D, western blot analysis of G9a function (C) and autophagy-related genes (D) in neuroblastoma cells expressing GFPsi or G9asi#4. α-Tubulin levels are shown as the loading control. Cells expressing GFPsi are shown as the biological control. E, neuroblastoma cells were plated at 1×103 cells per well in six-well culture plates. After 14 to 21 days of culture, soft agar colonies developed with cells expressing GFPsi. As shown, the cells with G9a knockdown were observed to give rise to small and scanty colonies in soft agar, Scale bars, 50 µm. F, Colonies that were larger than 0.5 mm or that contained more than 50 cells were recorded. Each column represents the average obtained from three independent experiments; error bars, SD. Statistical analysis was performed using two-tailed student's t-test, *p≤0.01.