Figure 4.

hESCs Avoid Anoikis by Exiting Their Undifferentiated State

(A) Quantification of Annexin V/NANOG double-positive hESCs treated for 24 hr with DMSO or FAKi. ^∗p < 0.05.

(B) Phase images of hESCs treated with DMSO or FAKi for 3 days. Scale bar, 100 μm.

(C) Immunofluorescence images of hESCs treated with DMSO or FAKi for 3 days. Cells were co-stained with DAPI and antibody against NANOG. Scale bar, 50 μm.

(D) Immunoblot of FAK, pSMAD2, and NANOG in hESCs treated with DMSO or FAKi for 3 days.

(E) Gene expression showing fold change for pluripotency-associated markers NANOG and OCT4 and early differentiation markers PAX6, SOX17, Goosecoid, and FOXA2 in hESCs treated with DMSO or FAKi for 3 days. ^∗p < 0.05 relative to DMSO.

(F) Gene expression fold change for pluripotency-associated and early differentiation markers [as in (E)] in hESCs treated with 10 μg/ml of MAB13 or IgG control for 3 days. ^∗p < 0.05, MAB13 relative to IgG and EBs relative to hESCs; n.s., not significant.

(G) Proposed model for FAK signaling in hESCs: ECM-integrin binding activates FAK, which induces PI3K upstream of AKT/MDM2 survival cascade leading to suppression of p53. In absence of FAK activity, the concomitant switch-off of AKT and elevation of p53 induces a caspase-dependent anoikis or downregulation of hESCs core genes and differentiation.

Data represent mean + SEM (n = 3 experiments). See also Figure S4.