Figure 2.

Inhibition of FAK Signaling Induces Cell Blebbing and a Caspase-Dependent Anoikis

(A) Quantification of Annexin V/7-AAD positive cells by flow cytometry in hESCs treated for 24 hr with the indicated concentrations of PF562271, DMSO, or untreated. ^∗p < 0.05 relative to DMSO.

(B) Caspase activity in hESCs treated for 5 hr with the indicated concentration of FAK inhibitors. ^∗p < 0.05 relative to DMSO.

(C) Caspase activity in hESCs treated for 5 hr with the indicated concentration of AKT inhibitors. ^∗p < 0.05 relative to DMSO.

(D) Cleaved caspase-3 expression in hESCs treated with DMSO, FAKi only, or with 50 μM ZVAD-FMK for 24 hr. ^∗p < 0.05 relative to DMSO.

(E) Phase images of hESCs treated for 24 hr with DMSO, FAKi only, or with Z-VAD-FMK. Inset shows blebbing in a single cell (a) or groups of cells (b). Scale bar, 100 μm.

(F) Dot plots of Annexin V/7-AAD-positive cells in hESCs treated for 24 hr with DMSO, FAKi only, or with Z-VAD-FMK.

(G) Immunoblot for FAK and GAPDH in hESCs nucleofected with mock control, control GFP vector (ctl vector) plus FAK siRNA (siFAK), or siFAK for 48 hr. Bottom: protein knockdown efficiency. ^∗p < 0.05.

(H) Top: phase images of hESCs after knockdown with β2-microglobulin siRNA (siB2M) control or siFAK for 66 hr. Bottom: hESCs after treatment with 10 μg/mL of IgG isotype or MAB13 antibody for 24 hr. Scale bars, 100 μm (top) and 50 μm (bottom).

(I) Quantification of Annexin V-positive hESCs after knockdown with siB2M or siFAK (66 hr). ^∗∗p < 0.03.

(J) Quantification of Annexin V/7-AAD-positive hESCs after knockdown with siB2M or siFAK (66 hr).

(K) Quantification of Annexin V/7-AAD-positive hESCs treated with IgG or MAB13 antibody (24 hr). ^∗p < 0.05.

Data represent mean + SEM (n = 3 experiments). See also Figure S2.