Fig. 4.

Pharmacologic ascorbic acid induces AIF1 release from mitochondria. (A) Top bar graph represents OCI-MY5-Scramble and -shRNA-AIF1 cells incubated with doxycycline to knockdown AIF1 for 2 days. Bottom bar graph represents EV OCI-MY5 and OE-AIF1 OCI-MY5 cells. All cells were treated with or without PAA at the specified concentrations. After 1 h treatment, PAA was washed away and cell viability was measured after 24 h. Knockdown and overexpression of AIF1 was confirmed by western blots. (B) Schematic representation of PAA inducing AIF1 cleavage, release and nuclear translocation in MM tumor cells. (C) OCI-MY5 WT cells with or without PAA. After 1 h PAA was washed away and cells were incubated with melphalan (Mel, 0–80 μM) for 4 h then lysed. AIF1, β-actin and γ-H2AX levels were analyzed by western blots. (D) OCI-MY5 WT cells were incubated with or without DFO (200 μM) for 3 h followed PAA (2 mM) treatment. After 1 h PAA was washed away and cells were lysed. AIF1 and β-actin levels were analyzed by western blots. White arrow represent AIF1 cleaved form. (E) Electron microscopy shows AIF1 immunolabeling stain of OCI-MY5 WT cells treated without (left) or with (right) PAA (2 mM). N, M, C respectively represent nucleus, mitochondria and cytoplasm. Blue arrows indicate the nuclear membrane and red arrowheads indicate AIF1 gold beads in cytoplasm or mitochondria. Black arrowheads indicate AIF1 gold beads in nuclei.