Fig 2: The roles of PI3K, AKT, and FoxO1 in 15d-PGJ2-induced NRROS expression are confirmed by transfection with respective siRNAs. RBA-1 cells were transfected with siRNA (a, d p85; b, e AKT, or c, f FoxO1) or scramble siRNA for 48 h and then incubated with 10 µM 15d-PGJ2 for 4 h (a–c) and for the indicated time intervals (d–f). The levels of NRROS, phospho-AKT, phospho-FoxO1, AKT, FoxO1, and GAPDH protein were analyzed by Western blotting. The data are presented as mean ± SEM, from three independent experiments (n = 3, number of independent cell culture preparations). #p < 0.05, as compared with the respective values significantly different as indicated

Fig 3: IGF-1/PI3K/Akt/FoxO1/GPR17 signaling. The expression of the GPR17 receptor on the cell membrane of SK-N-SH cells is regulated by IGF-1/PI3K/Akt/FoxO1 signaling. (a) In starved SK-N-SH cells, the expression of GPR17 receptor on the cell membrane is increased. This may be because the binding of FOXO1 to the promoter regions of GPR17 enhances GPR17 expression; (b) IGF-1 could promote the proliferation of SK-N-SH cells. This may be due to the action of IGF-1 receptor by IGF-1, followed by an increase in Akt phosphorylation (pAkt). pAkt promotes FOXO1 phosphorylation and reduces FOXO1 binding to the promoter regions of GPR17, thus resulting in the downregulation of GPR17 expression in starved SK-N-SH cells.

Fig 4: FoxO1 and Phospho-FoxO1 expression and nuclear translocation induced by KPC-exosomes in C2C12 myotubes. Differentiated C2C12 cells cultured in 25-cm2 culture-flasks with 5 ml DMEM were treated with KPC-exosomes (1 µg, 2 µg, 4 µg) for 24 h and 10-7 M insulin (5 min, 15 min, 30 min). (A) Total FoxO1 expression; expression in cytoplasm and nucleus. (B) Total Phospho-FoxO1 expression; expression in cytoplasm and nucleus. (C) Differentiated C2C12 cells cultured in 24-well plates were treated with 1 µg/ml KPC-exosomes for 24 h or 10-7 M insulin for 30 min. The nuclear (blue) translocation of FoxO1 (red) was verified by immunofluorescence. Scale bar, 20 µm. (D) 20 µg PKH67-dyed (green) exosomes in 100 µL PBS was injected through the caudal vein into the body of each mouse. The nuclear (blue) translocation of FoxO1 (red) was verified by immunofluorescence. Scale bar, 50 µm.

Fig 5: A model for the consequences of RB or PTEN deletion on E2F1/FOXO tumor suppressive apoptosis. RPCs with normal RB and PTEN protein levels do not proliferate due to minimal E2F activity. In the event of an RB mutation, E2Fs are deregulated and may complex with FOXO1 to induce apoptosis and suppress tumor emergence. Deleting PTEN activates p-AKT, causing p-FOXO1 inactivation and thereby disabling E2F-driven apoptosis, while allowing proliferation to occur. This model predicts that E2F and FOXO are simultaneously active in the nucleus only after certain oncogenic stresses, such as Rb1 loss.