Figure 4.

Treatment with N6-isopentenyladenosine (i⁶A) Induces Excessive Autophagy and Loss of GIC-Related Traits

(A) Representative images of immunostaining with an anti-ms²i⁶A antibody and MitoTracker in JKGIC2 cells. Note that the intracellular distribution of ms²i⁶A is limited to the mitochondria. Scale bars, 20 μm.

(B) Treatment with i⁶A, but not ms²i⁶A, for 24 h induces increases in LC3-II levels in JKGIC2 cells. GAPDH served as a loading control. The same results were reproduced three times.

(C) Treatment with i⁶A, but not ms²i⁶A, activates the autophagic program in JKGIC2 cells.

(D) Treatment with i⁶A for 24 h induces LC3 puncta formation in JKGIC2 cells.

(E) Quantification of primary spheres formed by JKGIC2 cells treated with i⁶A or ms²i⁶A. Treatment with i⁶A reduces the number of spheres, but this reduction is not rescued by the further addition of ms²i⁶A. Each bar represents the SD value from four independent replicates. *p < 0.05.

(F and G) Immunoblotting (F) and immunostaining (G) indicate that treatment with i⁶A, but not ms²i⁶A, decreases the protein levels of Nestin and Sox2 in JKGIC2 cells.

(H) Quantification of primary spheres formed by JKGIC2 cells (2,000/well) transfected with shRNAs against ATG5 and i⁶A. ATG5 knockdown successfully rescues the i⁶A-induced loss of stemness in JKGIC2 cells. Each bar represents the SD value from four independent replicates. *: p < 0.05.

(I and J) Exogenous transduction of murine Cdk5rap1 increases the amount of Nestin protein (I) and the number of spheres formed by JKGIC2 cells (2,000/well) (J). Each bar represents the SD value from four independent replicates. *p < 0.05.

(K) Ectopic expression of murine Cdk5rap1 prevents the loss of anchorage-independent growth ability in JKGIC2 due to exogenous treatment with 4 and 8 μM i⁶A. 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 S4.