Figure 6.

CDK5RAP1 Is Essential for Sustaining the GIC-Related Traits of GICs but Not That of Normal Brain Cells

(A) Left: Representative MR image and H&E staining of the brain tissue in a patient (case 1) with GBM. Tumor core and peritumor samples were harvested to extract RNA and nucleosides. Right: Mass spectrometry analysis of relative [i⁶A] shows that [i⁶A] is increased in the tumor core. Each bar represents the SD value from three independent replicates. *p < 0.05 versus the peritumor sample.

(B) Expression levels of CDK5RAP1 in human GBM specimens from the tumor core and peritumor areas are not significantly different. n = 5 per sample type.

(C) Expression level of VEGF in the peritumor and tumor core areas of patients with GBM. n = 5 per sample type.

(D) Mass spectrometry analysis of relative [ms²i⁶A] in the peritumor and tumor core areas of specimens from patients with GBM. Each bar represents the SD value from three independent replicates. *p < 0.05 versus the peritumor sample.

(E) Representative images of immunofluorescence analysis with anti-ms²i⁶A and anti-Nestin antibodies in the tumor core of patients with GBM. Note that the ms²i⁶A-positive cells and Nestin-positive cells overlap.

(F) Untreated NSCs, NSCs transformed with oncogenic lentiviruses, and mouse primary neurons transformed with oncogenic lentiviruses (all from Cdk5rap1^fl/fl mouse brains) either with or without transduced adenoviruses harboring Cre recombinase were subjected to the sphere formation assay. Transformed cells but not NSCs require Cdk5rap1 to sustain their anchorage-independent growth capacity. The data are presented as the number of spheres formed from 2,000 cells. Each bar represents the SD value from four independent replicates. *p < 0.05.

(G) Representative data of the gliosphere initiation assay. Primary astrocytes from wild-type or Cdk5rap1^fl/fl mouse brains were infected with Cre-inducible HRas^G12V-expressing pTomo lentiviruses. The cells were further infected with adenoviruses harboring Cre recombinase to activate HRas^G12V and recombine out the Cdk5rap1 alleles and were cultured in GIC medium for 7–10 days. The numbers of initiated spheres from 50,000 cells were counted. n = 4 per condition. For the procedure of the gliosphere initiation assay, see Figure S6. Cdk5rap1 is required to initiate the formation of gliospheres from primary astrocytes.

(H) Quantification of secondary, tertiary, and quaternary spheres formed by the transformed astrocytes harvested from the gliosphere initiation assay described in (G). Cdk5rap1 is required to sustain the self-renewal capacity of the transformed cells. The data are presented as the number of spheres formed from 2,000 cells. Each bar represents the SD value from four independent replicates. *p < 0.05.

Also see Figure S6.