Journal: Cell reports
Article Title: CDK5 Inhibition Resolves PKA/cAMP-Independent Activation of CREB1 Signaling in Glioma Stem Cells
Figure Lengend Snippet: CDK5 Regulates Self-Renewal and Asymmetric Cell Division in Drosophila Neuroblasts (A) Drosophila eye screen. (i) Drosophila normal eye. (ii) brat -RNAi using eyeless-GAL4 drives overgrowth of the eye. Using this phenotype, we performed a suppressor screen for kinases and found that reducing dcdk5 (v) and its activating partner, dp35 (iv), can reverse the overgrowth successfully. Suppression of two other kinases, dS6k (iii) and dEip63E (vi), did not revert the overgrowth completely. Graphs of the suppressor screen experiments show statistical significance. (B) Diagrams represent Drosophila P35 and Cdk5 are identical to human P35 ( CDK5R1 ) and human CDK5 by 66% and 79%, respectively. (C) brat -RNAi adult brain tumor shows high Mira expression (green) (i), indicating deregulated asymmetric division that generated these tumor stem cells. These cells also express high levels of active dCdk5 (white) (ii) compared with normal cells. RNAi suppression of dP35 and dCdk5 in brat -RNAi tumors reduced cell proliferation (PH3 staining, white) (iii, iv, and v) and the self-renewal marker (Mira, green) (vi, vii, and viii). Statistics are in Figure S1 . (D) We tested the potency of Cdk5 as an asymmetric cell division regulator by knocking down dCdk5 and by overexpressing human CDK5 specifically in neuroblasts. During asymmetric division, larval neuroblast mother cells (Mira positive, green) generate progenitors that are Mira negative and eventually differentiate (i, ii, and iii). Knock down of dCdk5 using RNAi specifically in neuroblasts reduced Mira expression without any visible change in daughter cells (red) (iv, v, and vi). However, ectopic expression of hCDK5 in neuroblasts disrupted the asymmetric division, generating more Mira-positive cells (vii, viii, and ix). Statistics are in Figure S1 . (E) CP681301, a specific CDK5 inhibitor, was fed to adult flies with brat -RNAi tumors. After 10 days of feeding, adult tumors lost expression of stem cell markers (Mira, green) (i and ii) and active phosphorylated dCdk5 expression (white) (iii and iv). (F) Suppressing dCdk5 activity with CP681301 increased the median survival of the brat -RNAi tumor-carrying flies by 2.85-fold. p
Article Snippet: In Vitro CP681301 Efficiency Test Thermo Fisher Scientific tested the inhibitory efficiency of CP681301 on active CDK5/P25 and CDK5/P35 in vitro .
Techniques: Expressing, Generated, Staining, Marker, Activity Assay