Figure 6.
Mitochondrial STAT6 accumulation under hypoxic condition induces cell death
(A and B) 293T cells were exposed to hypoxia (0.1%) for the indicated times. (A) Mitochondria and cytosol obtained from the 293T were subjected to Western blot. CYCS: mitochondrial marker. HSP90AB1: cytosolic marker.
(B) Western blot of immunoprecipitation using lysates from hypoxia-exposed 293T cells for indicated times.
(C) Representative confocal images of mitochondrial morphology and Cytochrome c release in empty vector- or STAT6-expressing U373-MG stable cell lines exposed to hypoxic condition for indicated times and stained with Cytochrome c and TOM20. Scale bar, 10μm.
(D) Mitochondria and cytosol obtained from STAT6 KO-293T cells transfected with Empty vector-, STAT6 WT, and ΔMTS exposed to hypoxia (0.1% O2, 24 h).
(E) Flow cytometry dotplots of annexin V and propidium iodide (PI) staining in Empty vector-, STAT6 WT, ΔSH2, and ΔMTS-expressing 293T exposed to hypoxic condition. Data are presented as means ± SD of 4 independent experiments. (∗p < 0.05).
(F and G) Clonogenic assay. Representative images of colonies are shown in left, and quantification of the number of colonies is depicted in right. 5 independent experiments (∗∗∗∗p < 0.0001), unpaired Student t-test (F) or One-way ANOVA with Scheffe’s post-hoc test (G).
(H and I) Soft-agar colony formation assay. Representative images of colonies are shown in left, and quantification of the number of colonies is depicted in right. 5 independent experiments (∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001), unpaired Student t-test (H) or One-way ANOVA with Scheffe’s post-hoc test; (I).