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. Author manuscript; available in PMC: 2017 Feb 8.
Published in final edited form as: Cancer Cell. 2016 Feb 8;29(2):201–213. doi: 10.1016/j.ccell.2016.01.005

Figure 2. Depletion of MLK4 attenuates a set of MES GSC phenotypes.

Figure 2

(A) Effects of MLK4 knockdown by shRNA (shMLK_1 or shMLK_2) and MLK4 knockout (KO) by CRISPR/Cas9 system on cell growth in MES 83 glioma spheres analyzed by AlamarBlue staining. Results were expressed as relative fluorescence units (RFU). Data are means ± SD (n = 6). ***p < 0.001.

(B) Representative images of EdU incorporation assays (left) and quantification of EdU positive cells (right) in MES 83 glioma spheres expressing shNT or shMLK4. Cells in green represent EdU positive cells. Nuclei were counterstained with DAPI (blue). Scale bar represents 20 μm. *p < 0.05.

(C) FACS plots of Annexin V and PI staining in MES 83 and PN 528 glioma spheres expressing shNT, shMLK4_1, or shMLK_2.

(D) Effects of or shMLK4_2 and MLK4 KO on sphere forming frequency of MES 83 glioma cells determined by limiting dilution assays. Stem cell frequency was calculated by ELDA analysis. Data are means ± SD (n = 18) ***p < 0.001. See also Figure S2.