Suppression of CaMKIIβ Inhibits ANO1-Mediated Glioblastoma Progression

Figures

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  Figure 1. The CaMKII blocker KN-93 decreases the migration and chloride currents of U251 cells. (A) Representative photographic images showing the migration of U251 cells treated with KN-93 as compared with the migration of the cells treated with dimethyl sulfoxide (DMSO). Scale bar, 200 μm. (B) The summary bar graph shows the reduced migration of U251 cells after treatment with KN-93 compared to that of cells treated with DMSO. (C) Averaged traces of whole-cell currents of U251 cells treated with KN-93. (D) The summary bar graph shows the inhibitory effect of KN-93 on the amplitude of the chloride current at ± 100 mV. Number on each bar indicates n for each condition. All values are mean ± s.e.m. P-values were obtained with Student’s t-test. * p < 0.05, ** p < 0.01, and *** p < 0.001.

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  Figure 2. KN-93 reduces the surface expression and activity of ANO1 in U251 cells. (A) U251 cells treated with DMSO or KN-93 were imaged using antibodies against ANO1 and WGA647 (WGA), a plasma membrane marker. Scale bar, 20 μm. (B) The Pearson’s correlation coefficient for ANO1 with KN-93 was significantly less than the value obtained for ANO1 with DMSO in U251 cells. (C) Cell surface biotinylation results from membrane protein fractions from U251 cells treated with DMSO or KN-93. (D) The summary bar graph showing data obtained from three independent experiments as in (C). (E) Averaged traces of whole-cell currents of U251 cells treated with DMSO or T16Ainh-A01, an ANO1 inhibitor. (F) The summary bar graph shows the inhibitory effect of KN93 or T16Ainh-A01 on ANO1 current amplitude at ±100 mV. (G) Averaged traces of normalized T16Ainh-A01-sensitive currents of U251 cells treated with DMSO or KN-93. (H) The bar graph shows normalized T16Ainh-A01-sensitive current densities (G) at + 100 mV. Number on each bar indicates n for each condition. All values are mean ± s.e.m. P-values were obtained with Student’s t-test. * p < 0.05, ** p < 0.01, and *** p < 0.001. n.s means not significant.

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  Figure 3. CaMKIIβ specifically increases the surface expression and activity of ANO1 in U251 cells. (A) The mRNA expression of CaMKII isoforms in U251 cells were detected with specific primers for CaMKIIα, β, γ and δ. (B) Normalized expression of CaMKII isoforms as compared to that of GAPDH in U251 cells. (C) Effect of CaMKII isoform overexpression on ANO1-mediated whole cell chloride currents in U251 cells. (D) The summary bar graph showing ANO1-mediated current density at ± 100 mV. (E) Cell surface biotinylation results from membrane protein fractions from U251 cells transfected with mCh-CaMKIIα and mCh-CaMKIIβ. (F) The summary bar graph showing data obtained from at least three independent experiments as in (E). Number on each bar indicates n for each condition. All values are mean ± s.e.m. P-values were obtained with Student’s t-test. ** p < 0.01 and *** p < 0.001. n.s means not significant.

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  Figure 4. CaMKIIβ knockdown reduces the surface expression of ANO1 in U251 cells. (A,B) Validation of the silencing efficiency of the siRNA against CaMKIIβ using qPCR and Western blotting. (C) U251 cells transfected with Sc shRNA or CaMKIIβ siRNA were imaged using antibodies against ANO1 and WGA. Nuclei were stained using DAPI staining solution. Scale bar, 20 μm. (D) Cell surface biotinylation results from U251 cells transfected with Sc shRNA or CaMKIIβ siRNA. (E) The summary bar graph shows the summary of (D), data obtained from three independent experiments. (F) Averaged traces of whole-cell currents of U251 cells transfected with Sc shRNA, CaMKIIβ siRNA, or T16Ainh-A01 (A01). (G) The summary bar graph shows the inhibitory effect of CaMKIIβ siRNA on ANO1–mediated current amplitude at ±100 mV. The bar graph shows normalized A01-sensitive current densities at + 100 mV. Number on each bar indicates n for each condition. All values are mean ± s.e.m. p-values were obtained with Student’s t-test. * p < 0.05 and ** p < 0.01.

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  Figure 5. Gene silencing of ANO1 or CaMKIIβ leads to the attenuation of migration and invasion in U251 cells. (A,B) Validation of the silencing efficiency of the shRNA against ANO1 using qPCR and Western blotting. (C) Cell invasion assay of U251 cells infected with Lenti-ANO1 shRNA and transfected with CaMKIIβ siRNA. (D) The averaged bar graph shows the summary of (C), data obtained from three independent experiments. (E) Representative photographic images of the migration of U251 cells infected with Lenti-ANO1 shRNA and transfected with CaMKIIβ siRNA and the cells with Lenti-Sc shRNA and Sc siRNA control. Scale bar, 100 μm. (F) The averaged bar graph shows the summary of (E), data obtained from three independent experiments. All values are mean ± s.e.m. p-values were obtained with Student’s t-test. * p < 0.05, ** p < 0.01, and *** p < 0.001.

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  Figure 6. Inhibition of CaMKIIβ and/or ANO1 decreased migration and invasion of U87MG cells. (A,B) Validation of the silencing efficiency of the Lenti-ANO1shRNA and the CaMKIIβ siRNA with qPCR and Western blot in U87MG cells. (C) Cell invasion assay of U87MG cells infected with Lenti-ANO1shRNA and transfected with CaMKIIβ siRNA. (D) The averaged bar graph shows the summary of (C), data obtained from three independent experiments. (E) Representative photographic images of the migration of U87MG cells infected with Lenti-ANO1shRNA and transfected with CaMKIIβ siRNA as compared to the cells with Lenti-Sc shRNA and Sc siRNA control. Scale bar, 100 μm. (F) The averaged bar graph shows the summary of (E), data obtained from three independent experiments. All values are mean ± s.e.m. p-values were obtained with Student’s t-test. * p < 0.05, ** p < 0.01, and *** p < 0.001.