Figure 4
From: Wee1 inhibition potentiates Wip1-dependent p53-negative tumor cell death during chemotherapy
A caspase-3-dependent apoptosis correlated to mitochondrial superoxide production following cisplatin-induced DNA damage is Wip1-dependent in Saos2 osteosarcoma cells, and is related to Hipk2 deregulation by Wee1 kinase inhibition. (a) Anti-active caspase-3 flow cytometry assay following a treatment of Saos2 osteosarcoma cells with 25 μM CDDP alone, with 1 μg/ml doxycycline to induce Wip1 overexpression, or in triple combination with 75 nM MK-1775. (b) Flow cytometry profile showing the Wip1-dependency, induced by doxycycline, of mitochondrial ROS formation after a treatment with 25 μM of CDDP for 24 h. (c) Flow cytometry assessment of mitochondrial superoxide production. Saos2 osteosarcoma cells were treated with increasing doses of CDDP for 24 h with or without induction of Wip1 by 1 μg/ml doxycycline. Mitochondrial superoxide formation has been detected using MitoSOX superoxide indicator by flow cytometry after an incubation of cells with 2 μM of the dye for 15 min at 37 °C. (d) Anti-γH2AX flow cytometry assay following 28 h treatment of Saos2 osteosarcoma cells with 25 μM CDDP, 75 nM MK-1775 and 1 μg/ml doxycycline to induce Wip1 overexpression. (e) Western blot of anti-Hipk2 with or without a 25 μM CDDP treatment and a Wee1 partial inhibition by 75 nM of MK-1775 for 20 h
