Figure 1.
Phorbol myristate acetate (PMA) and ionomycin (Io) inhibit glioblastoma multiforme (GBM) cell proliferation.
Proliferation was assessed by MTT assay and cell counting. The cells were plated in triplicate, treated with 50/mL PMA and/or 10 ng/mL Io, and analyzed for proliferation for 120 h in MTT assay and for 48 h in cell counting. The graphs are representatives of three independent experiments. (A, B) PMA and/or Io treatment significantly inhibited U87 cell proliferation. (C, D) PMA and/or Io also markedly suppressed the proliferation of U251 cells. *P<0.05 (compared with the control).
Figure 2.
PMA and Io induce GBM cell apoptosis.
Apoptotic cells were examined 48/or Io treatment in U87 and U251 cells. (A, B) Quantitative analysis of the apoptotic cells after treatment with PMA and/or Io; apoptotic cells were determined by the TUNEL assay. The graphs are representative of three independent experiments. (C–H) Representative photomicrograph (5000×) of U87 cells (C–E) and U251 cells (F–H) using transmission electron microscopy. In the control groups (C, F), the nuclear shape is normal and the nucleolus is clear. In contrast, nuclear shape changes, and condensation of chromatin and nuclear fragmentation can be observed in treated cells (D, E, G, H). *P<0.05 (compared with the control).
Figure 3.
PMA and Io induce cell cycle arrest and apoptosis in U87 GBM cells.
U87 cells were stained with propidium iodide (PI) and analyzed by flow cytometry for cell cycle and death. The graphs are representative of three independent experiments. (A, B) Cell cycle was analyzed by the incorporation of PI. The cells were plated in triplicate and analyzed 24 h after treatment. The percentage of cells in each phase of the cell cycle is indicated in the graphs. PMA and Io significantly arrested cells at G0/G1 phase. (C, D) Analysis of cell death 48 h after treatment. The percentage of cells in sub-G0 is shown in the graph.
Figure 4.
The mRNA expression of NFAT1 and Fas obtained from microarray analysis in 111 clinical samples was analyzed by cluster analysis and Pearson correlation analysis.
(A) NFAT1 was overexpressed in high-grade gliomas. (A, B) The expression of NFAT1 significantly correlated with the expression of Fas in gliomas. (C) Western bot showed that NFAT1 was highly expressed in GBM clinical samples as well as in U87 and U251 cells, compared with low-grade glioma and normal brain tissue samples. The graph is a representative of three independent experiments.
Figure 5.
NFAT1 can be activated by PMA and Io in GBM cells.
The graphs are representative of three independent experiments. (A, B) Immunofluorescent staining demonstrated that in U87 and U251 cells, NFAT1 is hyper-activated by PMA and Io treatment. Treatment of U87 and U251 cells with PMA and/or Io for 60 min caused rapid activation and nuclear translocation of NFAT1. (C, D) Semi-quantification of the mean intensity of NFAT1 fluorescence in the nucleus of U87 (C) and U251 (D) cells using Image pro plus software. *P<0.05 (compared with the control U87 and U251 cells respectively).
Figure 6.
NFAT1 down-regulation prevents PMA and Io-induced cell cycle arrest and cell death.
The graphs are representative of three independent experiments. (A) The expression of NFAT1 was effectively knocked down in U87 cells by transfection of specific small hairpin RNA. Cell counting (B) and MTT assay (C) showed that the knockdown of NFAT1 prevented the inhibition of proliferation by PMA and Io. (D–G) Cells were stained with PI and analyzed by flow cytometry for cell cycle and death. (D, E) Cell cycle was analyzed by the incorporation of PI. The cells were plated in triplicate and analyzed 24 h after treatment. The percentage of cells in each phase of the cell cycle is indicated in the graphs. Down-regulation of NFAT1 markedly prevented cell cycle arrest induced by PMA and Io. (F, G) Analysis of cell death 48 h after treatment. The percentage of cells in sub-G0 is shown in the graph. Silence of NFAT1 also prevented PMA and Io-induced U87 cell death. *P<0.05 (compared with the U87-control-shRNA cells).
Figure 7.
PMA and Io-induced apoptosis is NFAT1-dependent in U251 cells.
The graphs are representative of three independent experiments. (A) The expression of NFAT1 was effectively knocked down in U251 cells by transfection of specific small hairpin RNA. (B) The knockdown of NFAT1 prevented the inhibition of proliferation by PMA and Io in U251 cells. (C) The TUNEL assay showed that NFAT1-silencing inhibited the apoptosis induced by PMA and Io. (D) The induction of FasL by PMA/Io was NFAT1-dependent. (E) The TUNEL assay showed that FasL neutralization by a specific anti-FasL antibody significantly inhibited PMA and Io-induced apoptosis in U251 cells. *P<0.05 (compared with the control respectively).
Figure 8.
PMA and Io-induced apoptosis is NFAT1-dependent in U87 cells.
The graphs are representative of three independent experiments. (A) The TUNEL assay showed that NFAT1-silencing inhibited the apoptosis induced by PMA and Io. (B) Real-time PCR and western blot analysis demonstrated that treatment of PMA and Io elevated the expression of Fas and FasL. (C) However, the induction of FasL was NFAT1-dependent. (D) The TUNEL assay showed that FasL neutralization by a specific anti-FasL antibody significantly inhibited PMA and Io-induced apoptosis. *P<0.05 (compared with the control respectively).