Figure 1.
The establishment of stable LN229 cells expressing AQP4 siRNA plasmid.
A. Western blotting analysis of AQP4 expression in parental LN229, scr/LN229 and siAQP4/LN229 clone 1, clone 2, clone 3 and clone 4 cells. The expression of AQP1 in parental LN229 and siAQP4/LN229 clone 2 cells were also detected, the kidney and normal brain tissue were used as positive controls, β-actin was used as a loading control. B. Cells osmotic fragility was examined after AQP4 reduction. The survival rate of the control was 71.61% ± 11.75% (mean ± SD), whereas it decreased to 29.46% ± 14.84% (mean ± SD) in siAQP4/LN229 clone 2 cells. C. Comparison of cell proliferation in scr/LN229 and siAQP4/LN229 clone 2 cells by MTT assay. Each data point was an average of triplicate assays (Bars, standard deviation; two-way ANOVA analysis, **P<0.01).
Figure 2.
Colony formation of LN229 cells after AQP4 reduction.
A. representative images of colony formation for scr/LN229 and siAQP4/LN229 clone 2 cells (200×). The images were taken at the 10th day of the assay. B. Quantitation of colony number in two groups (scr/LN2229 cells group and siAQP4/LN229 clone 2 cells group). Results were from images (100×) of three independent experiments. C. Quantitation of colony size in two groups (scr/LN2229 cells group and siAQP4/LN229 clone 2 cells group). The data was collected by representative images from three repeated experiments (200×) (two-way ANOVA analysis, **p<0.01).
Figure 3.
Reduction of AQP4 induced apoptosis of LN229 cells.
A. Images showed DAPI staining of LN229, scr/LN229 and siAQP4/LN229 clone 2 cells (200×). LN229 and scr/LN229 groups were regarded as control. The quantitative result was shown by the histograms (**P<0.01). Quantitative results were analyzed (two-way ANOVA analysis, **P<0.01). B. In order to detect the Cyt-C efflux from mitochondria, the cells were incubated with anti-Cyt-C antibody at room temperature for 60 min in the dark, and then the content of Cyt-C was detected by flow cytometry. The quantitative result was shown by the histograms (**P<0.01).
Figure 4.
Reduction of AQP4 induced apoptosis of LN229 cells.
A. Protein level of Cyt-C was determined by Western blotting. Expression of Cyt-C was quantitated by densitometry and normalized to β-actin expression. Results were analyzed using two-way ANOVA analysis, **P<0.01. Western blotting results showed a representative blot taken from three independent experiments. B. Annexin V Apoptosis Kit was used to detect cells apoptosis. Flow cytometric analyses of scr/LN229 and siAQP4/LN229 clone 2 cells were shown. Apoptosis rate in scr/LN229 cells and in siAQP4/LN229 clone 2 cells were (3.8±0.71)% and (8.28±1.25)%, respectively.
Figure 5.
The expression of Bad and Bcl-2 in LN229 cells were detected by Western blotting after AQP4 reduction.
Western blotting was performed by using either anti-Bad antibody or anti-Bcl-2 antibody. Expression of Bad and Bcl-2 were quantitated by densitometry and normalized to β-actin expression. Data were analyzed using two-way ANOVA analysis **P<0.01.
Figure 6.
Reduction of AQP4 by PMA in LN229 cells induced apoptosis.
A. The expression of AQP4 in LN229 cells with PMA (0 µM, 1 µM, 5 µM) treatment (24 hr) was detected by Western blotting. B. Western blotting was performed by using anti-Cyt-C, anti-Bad or anti-Bcl-2 antibody. (5 µM of PMA was used.) Expression of Cyt-C, Bad and Bcl-2 were quantitated by densitometry and normalized to β-actin expression. Data were analyzed using two-way ANOVA analysis **P<0.01.
Figure 7.
Annexin V Apoptosis Kit was used to detect cells apoptosis of U87 cells with transient transfection of AQP4 siRNA.
A. Western blotting analysis of AQP4 expression in scr/U87 and siAQP4/U87 cells. B. Flow cytometric analysis of U87 cells with transient transfection of AQP4 siRNA by Annexin V Apoptosis Kit. Apoptosis rate in scr/U87 cells was (0.81±0.11)% and it was (16.07±2.43)% in siAQP4/U87 cells. Data was representative of three independent experiments. Data were analyzed using two-way ANOVA analysis **P<0.01.
Figure 8.
Reduction of AQP4 induced glioma cells apoptosis in in vivo assay.
(A) Stable clones of scr/LN229 and siAQP4/LN229 were subcutaneous injected into Nu/Nu mice respectively. The size of tumors was measured each week. 6 weeks later, the representative images of tumor size in each group were captured. (B) The results of tumor volume in vivo were analyzed by two-way ANOVA analysis, **P<0.01.
Figure 9.
The expression of Bad, Cyt-C and Ki67 in xenograft tumor tissues.
Xenograft tumor tissues were obtained from subcutaneously inoculated Nu/Nu mice which were sectioned for immunohistochemistry to detect the expression of Bad, Cyt-C and Ki67. A1, B1 and C1 were images of scr/LN229 group, A2, B2 and C2 were images of siAQP4/LN229 clone 2 group (400×).
Table 1.
The expression of Bad,Cyt-C and Ki67 in xenografts.