Contribution of the PTEN/PI3K/Akt axis to H₂O₂-induced apoptosis in C-kit⁺ CSCs.

<p>(a) Flow cytometry was performed to detect apoptosis using Annexin V-FITC/PI staining in C-kit⁺ CSCs that underwent different treatments. The first quadrant (%f Gated) shows the necrotic cells (Annexin V- /PI+), The second quadrant (% Gated) shows the late apoptotic cells(Annexin V+/PI+), The third quadrant (% Gated) shows the live cells (Annexin V- /PI-)and The fourth quadrant (% Gated) shows the early apoptotic cells(Annexin V+/PI -), PI = propidium iodide.(b)The percentage of apoptotic cells was representing as both early and late apoptotic cells. (n = 3, *P<0.05 versus the H₂O₂ group. ^# P<0.05 versus the H-Exo group). (c) Representative immunofluorescence staining of Hoechst33258 (blue), TUNEL (green) and merged images. Photos were taken randomly using fluorescence microscopy. Scale bar: 20 μm. (d) The panel shows the percentages of TUNEL positive cells. (n = 6, *P<0.05 versus the H₂O₂ group. ^# P<0.05 versus the H-Exo group). (e) Representative immunofluorescence staining of DAPI(blue), Cleaved-Caspase-3 (red) and merged images. Photos were taken randomly using fluorescence microscopy. Scale bar: 20 μm. (f-g)Apoptotic was further confirmed by immunoblotting for cleaved-caspase-3, (n = 3, *P<0.05 versus the H₂O₂ group. ^# P<0.05 versus the H-Exo group).