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1.
Figure 7.

Figure 7. From: Voltage-gated potassium channel EAG2 controls mitotic entry and tumor growth in medulloblastoma via regulating cell volume dynamics.

Model of EAG2's regulation on MB growth. (A) EAG2 protein is normally expressed at modest levels in the adult cerebellum and is significantly up-regulated in a subset of MBs. (B) EAG2 confers a growth advantage to MB cells by promoting cell cycle progression through the regulation of PMC, the abrogation of which leads to strong mitotic defects associated with hyperactivation of the p38 MAPK tumor suppressor pathway. Ectopic membrane expression of EAG2 in nontumor cells results in strong cell volume reduction, the formation of a mitotic-like morphology, defective cell cycle progression, and apoptosis.

Xi Huang, et al. Genes Dev. 2012 August 15;26(16):1780-1796.
2.
Figure 2.

Figure 2. From: Voltage-gated potassium channel EAG2 controls mitotic entry and tumor growth in medulloblastoma via regulating cell volume dynamics.

EAG2 up-regulation is a hallmark in subset of human MBs. (A) EAG2 transcript levels are significantly up-regulated in the tumor biopsies in 11 of the 12 MB patients. (B) mRNA microarray analyses in a cohort of 103 MB patient tumor tissues identify significant EAG2 up-regulation in 14% (14 of 103) of MBs with different molecular and histological subgroups, age, and sex. (C) In previously microarrayed mouse MBs (Gibson et al. 2010), Eag2 increases in a subset of both Shh- and Wnt-driven MBs. (D) Immunohistochemical analyses of EAG2 expression in 35 paraffin-embedded human MB tissues show differential expression patterns. Bar, D, 20 μm.

Xi Huang, et al. Genes Dev. 2012 August 15;26(16):1780-1796.
3.
Figure 5.

Figure 5. From: Voltage-gated potassium channel EAG2 controls mitotic entry and tumor growth in medulloblastoma via regulating cell volume dynamics.

EAG2 knockdown results in defective PMC, with cell volume increase at late G2 phase, associated with activation of the tumor suppressor p38 MAPK pathway. (A) Cell volume measurements of soluble GFP-filled Vandy-MB-11 cells. GFP expression in Vandy-MB-11 cells is achieved by infection with GFP-expressing lentivirus. (B) EAG2-depleted cells display robust activation of the p38 MAPK pathway. The ERK pathway is modestly regulated by EAG2 function and p38 inhibition. SB203580 normalizes the DNA damage in EAG2-depleted MB cells. (C,D) The p38 MAPK inhibitor SB203580 rescues EAG2-depleted Vandy-MB-11 cell growth phenotype in a dose-dependent manner. (E) SB203580 promotes G2–M transition after EAG2 knockdown. (F) Following EAG2 knockdown, Vandy-MB-11 xenografted tumors display hyperactivation of the p38 MAPK pathway with prominent phospho-p38 staining and a significantly decreased percentage of cells stained with phospho-histone 3 and with condensed chromosomes, indicating defective mitotic entry in vivo (N = 3 each for control and EAG2 knockdown tumors). Bars: A, 20 μm; F, 15 μm.

Xi Huang, et al. Genes Dev. 2012 August 15;26(16):1780-1796.
4.
Figure 6.

Figure 6. From: Voltage-gated potassium channel EAG2 controls mitotic entry and tumor growth in medulloblastoma via regulating cell volume dynamics.

Ectopic EAG2 expression leads to cell volume reduction and cell cycle defects. (A) Cell volume measurements of soluble GFP-filled COS7 and HEK293 cells. COS7 and HEK293 cells are transfected with GFP, GFP + EAG2, or GFP + Kv4.2. Overexpression of EAG2, but not Kv4.2, strongly reduces COS7 and HEK293 cell volumes. The COS7 and HEK293 cells are at interphase, as evidenced by their lack of phospho-histone 3 immunoreactivity. Note that prominent ectopic retraction fibers are seen only in EAG2 transfected cells (white arrow). (B) Overexpression of EAG2, but not Kv4.2, leads to a profound growth phenotype with defective cell cycle and apoptosis. White-field images of comparably seeded COS7 and HEK293 show that EAG2 overexpression reduces the overall cell growth. EAG2 overexpression leads to a significant reduction of cells at G1 and G2/M phases as well as a marked increase of apoptotic cell death, as evidenced by the increase of cells at the sub-G1 phase and Caspase 3/7 activity. (C) Transfected EAG2 localizes to the plasma membrane marked by farnesylated eGFP. Both the whole-cell three-dimensional stacked and single optical slice views of a typical EAG2-overexpressing COS7 cell are shown. Note the prominent plasma membrane localization of EAG2 (yellow arrows). Bars: A, 20 μm; B, 10 μm.

Xi Huang, et al. Genes Dev. 2012 August 15;26(16):1780-1796.
5.
Figure 3.

Figure 3. From: Voltage-gated potassium channel EAG2 controls mitotic entry and tumor growth in medulloblastoma via regulating cell volume dynamics.

RNAi knockdown of EAG2 impairs MB cell growth and tumorigenicity. (A) Representative images of comparably seeded DAOY cells, low-passage Vandy-MB-11 cells, and PtchLacZ/+; p53−/− mouse MB cells treated with scrambled/EAG2 shRNA for 7–10 d. The clonogenic capacity of EAG2 knockdown cells is dramatically impaired compared with the control cells. EAG2 RNAi knockdowns effectively reduce endogenous EAG2 protein expression. (B) Noninvasive bioluminescence imaging (BLI) shows significantly reduced tumor burden in the mice injected with EAG2-depleted Vandy-MB-11 cells. Note the presence of spinal cord metastases (red arrows) in the six out of nine (67%) mice of only the control group. Histological analyses in another cohort of xenografted mice show that the tumors with EAG2 knockdown are much smaller in size. (C) While control tumors show steady growth rate, the EAG2 knockdown tumors display much slowed growth as measured by consecutive BLI measurements in Vandy-MB-11 xenografts. (D) Kaplan-Meier survival analyses show that EAG2 knockdown in tumors significantly improves survival in the Vandy-MB-11 xenografted mice. (E) While control tumors show a steady growth rate, the EAG2 knockdown tumors display much slower growth as measured by consecutive BLI measurements in the DAOY xenografts. (F) Kaplan-Meier survival analyses show that EAG2 knockdown in tumors significantly improves survival in the DAOY xenografted mice. (**) P < 0.005; (*) P < 0.05 by Student's t-test. Bar, A, 25 μm.

Xi Huang, et al. Genes Dev. 2012 August 15;26(16):1780-1796.
6.
Figure 1.

Figure 1. From: Voltage-gated potassium channel EAG2 controls mitotic entry and tumor growth in medulloblastoma via regulating cell volume dynamics.

Eag2 is highly overexpressed in Shh-driven mouse MBs. (A) Microarray analyses comparing transcript levels of several potassium channels between MBs and control cerebellum. Shh pathway activation is confirmed by the increased Gli1 and Mycn expression. (B) Relative ion channel gene expression levels in the PtchLacZ/+ MB and control cerebellum. (C) Conventional RT–PCRs demonstrate significantly increased Eag2 transcript levels in tumors compared with the normal cerebella of different developmental stages. (D) Quantitative RT–PCRs demonstrate significantly increased Eag2 transcript levels in tumors compared with the normal cerebella of different developmental stages (N = 3 for each stage). (E) Eag2 RNA in situ hybridization shows moderate expression in the adult cerebellar molecular layer (ML), purkinje layer (PL), and internal granule neuron layer (GL) and dramatic up-regulation in the Math1-Cre; SmoM2 MB tumor (T) tissue but not its adjacent nontumor (NT) tissue. (F) Western blotting shows a marked increase of Eag2 protein in the Math1-Cre; SmoM2 MB tumors (N = 3) compared with the age-matched control cerebella (N = 3). (G) Eag2 immunostaining shows prominent expression in the MB tumor tissue. (H) Whole-cell voltage clamp recordings of mouse MB cells ex vivo. A representative snapshot of electrode patching a randomly selected MB cell in an acute tumor slice is shown. Whole-cell voltage clamp traces from a MB cell in an acute tumor slice reveal a prominent outward current, a substantial portion of which is sensitive to the EAG2 blocker astemizole. Cells were stepped from a holding potential of −80 mV to −40 mV to +80 mV in 40-mV steps. Application of astemizole effectively reduces the outward conductance in four out of four MB cells tested. (**) P < 0.001 by Student's t-test. Bars: E, 250 μm; G, 25 μm.

Xi Huang, et al. Genes Dev. 2012 August 15;26(16):1780-1796.
7.
Figure 4.

Figure 4. From: Voltage-gated potassium channel EAG2 controls mitotic entry and tumor growth in medulloblastoma via regulating cell volume dynamics.

Dynamic EAG2 protein localization and profound mitotic phenotype associated with EAG2 knockdown. (A) EAG2 localizes within the intracellular compartments of the Vandy-MB-11 cells during interphase, as evidenced by the lack of immunostaining signal in the cell periphery. EAG2 protein can be detected on the cell surface, outlined by cortical F-actin in Vandy-MB-11 cells at late G2 phase and mitosis. (B) The top left panels show representative images of whole-cell recording from DAOY cells during interphase and mitosis. The top right traces show representative whole-cell voltage clamp traces from mitotic DAOY cells, revealing a prominent slowly activating outward current. Cells were voltage-clamped at a holding potential of −80 mV, then stepped from −60 mV to +100 mV in 40-mV steps. Dividing DAOY cells display significantly greater outward potassium current both in absolute amplitude (bottom left) and in terms of current density (bottom right). (*) P < 0.01 by Mann-Whitney U-test. (C) FACS analyses of Vandy-MB-11 cells treated with control and EAG2 shRNA show a significant increase in the proportion of cells in the G2/M phase following EAG2 knockdown. (D) Phospho-histone 3 staining shows significantly fewer cells with strong immunopositivity on condensed chromosomes and more cells with weak, diffuse nuclear immunopositivity after EAG2 knockdown. (E) Time-lapse microscopy on Vandy-MB-11 cell mitosis (in minutes). (F) Quantification of mitotic duration. (G) Relative Caspase 3/7 activity in Vandy-MB-11 cells. (H) Western blotting shows reduced EAG2 and Cyclin B1 expression after EAG2 knockdown, while Cyclin A, Cyclin D1, Cyclin E1, Cdk1, and phospho-Cdk1 expression is comparable between knockdown and controls. (I) Western blot showing decreased DNA damage and DNA damage response (DDR) pathway activation and comparable p53 phosphorylation in the EAG2-depleted Vandy-MB-11 cells compared with the control cells. (J) Western blot showing comparable level of phospho-AKT protein in knockdown and control cells. Bars: A,E, 10 μm; D, 20 μm.

Xi Huang, et al. Genes Dev. 2012 August 15;26(16):1780-1796.

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