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. 2019 Oct 8;21:42–56. doi: 10.1016/j.isci.2019.10.012

Figure 1.

Figure 1

CDK5RAP1 Is Required to Sustain GIC-Related Traits

(A) Left: Quantification of primary and secondary spheres formed by JKGIC2-shControl, JKGIC2-shCDK5RAP1#1, JKGIC2-shCDK5RAP1#2 and JKGIC5-shControl, JKGIC5-shCDK5RAP1#1, JKGIC5-shCDK5RAP1#2 cells. CDK5RAP1 is required to sustain the anchorage-independent growth capacity of GICs. The data are presented as the number of spheres formed from 2,000 seeded cells. Each bar represents the SD value of four independent replicates. *p < 0.05. Sequence information of shRNAs is show in Table S1. Right: Representative phase contrast images of these cells. Scale bars, 100 μm.

(B) Quantification of primary spheres initiated from the single cells of JKGIC2-shControl, JKGIC2-shCDK5RAP1#1, JKGIC2-shCDK5RAP1#2, JKGIC2-shCDK5RAP1#5 and JKGIC5-shControl, JKGIC5-shCDK5RAP1#1, JKGIC5-shCDK5RAP1#2, JKGIC5-shCDK5RAP1#5. CDK5RAP1 is required for sustaining the sphere formation capacity of GICs. The data are presented as the percentage of spheres formed from 48 wells of single cells. Each bar represents the SD value of four independent replicates. *p < 0.05.

(C) Left: Representative immunoblotting images of GIC markers in shControl- and shCDK5RAP1-transfected JKGIC2 cells. Sox2, Olig2, POU3F2, and SALL2 levels were determined 4 days after infection with respective shRNA-encoding lentivirus. GAPDH served as a loading control. The experiment was repeated three times. The information of antibodies used in the present study is shown in Table S2. Right: Relative expression levels of CDK5RAP1 4 days after the lentiviral transduction of shRNAs. Each bar represents the SD value from three independent replicates. Sequence information of the primers for qPCR is shown in Table S1.

(D) Representative immunostaining images of JKGIC2-shControl and JKGIC2-shCDK5RAP1 cells. Transfection with shCDK5RAP1 reduced the number of Sox2-and Nestin-positive cells. Scale bars, 20 μm.

(E–G) CDK5RAP1 is required to sustain the tumorigenic potential of GICs. After shRNA induction, JKGIC1, JKGIC2, and JKGIC5 cells were subcutaneously injected (E, n = 3 per condition) and JKGIC2 were intracranially (F, n = 5 per condition) injected. Notably, CDK5RAP1 knockdown prolonged the overall survival of the mice with brain tumors (G). *p < 0.05. Scale bars: 1 cm in (E) and 2 mm in (F).

Also see Figure S1.