Joo Young Kim Su-Jin Yu Hyun Ju Oh Ji Young Lee Yongjin Kim Jeongwon Sohn

Journal Name: Apoptosis

Panaxydol induces apoptosis through an increased intracellular calcium level, activation of JNK and p38 MAPK and NADPH oxidase-dependent generation of reactive oxygen species

Joo Young Kim· Su-Jin Yu· Hyun Ju Oh · Ji Young Lee · Yongjin Kim · Jeongwon Sohn

Corresponding author: Jeongwon Sohn, Department of Biochemistry, Korea University College of Medicine and Korean Institute of Molecular Medicine and Nutrition, Seoul 136-705, Korea. Tel: 82-2-920-6192; Fax: 82-2-923-0480; E-mail:

SupplementaryFigures

Supplementary Fig. 1. Overexpression of Bcl-2 protein in stably transfected cells. MCF-7 and Jurkat cells were stably transfected with the bcl-2 cDNA or an empty vector pcDNA3 as described in Materials and methods. Protein expression of Bcl-2 was analyzed by immunoblotting.

Supplementary Fig. 2. Loss of mitochondrial membrane potential by panaxydol treatment. Cells wereloaded with 10 μM JC-1(Invitrogen) for 30 min prior topanaxydol treatment. MCF-7 and Jurkat cells were treated with 40 and 30 g/ml of panaxydol, respectively. MCF-7 cells were subjected to fluorescence microscopy at indicated time points, whereas Jurkat cells were subjected to a flow cytometry analysis 5 h after panaxydol treatment at different concentrations. P indicates panaxydol.

Supplementary Fig. 3. Suppression of mitochondrial ROS generation by NADPH oxidase inhibitors in Jurkat cells. aMitochondrial ROS generation by panaxydol treatment. Cells were loaded with MitoSOX Red for 30 min, and after panaxydol treatment (40 g/ml), mitochondrial ROS levels were assessed by flow cytometry at indicated time points. bCells were pretreated with DPI (10 M) or apocynin (300 M) for 1 h, and mitochondrial ROS levels were measured 45 min after panaxydol treatment (40 g/ml). P indicates panaxydol.

Supplementary Fig.4. Panaxydol-induced cell death is inhibited by silencing Nox2 and Nox4 expression. MCF-7 cells were transfected with Nox2 and Nox4 siRNAs and treated with panaxydol 48 h after transfection. aCell death after panaxydol treatment (50 g/ml) was analyzed by PI staining and fluorescenct microscopy. P indicates panaxydol. bmRNA expression of Nox2 and Nox4 was analyzed by RT-PCR 48 h after siRNA transfection.

Supplementary Fig.5. Panaxydol-induced increase in [Ca2+]i is not blocked by NADPH oxidase inhibitors. aMCF-7 cells were pretreated with DPI (10 M) for 1 h, and simultaneously loaded with fura-2 AM for the last 30 min. Changes in [Ca2+]i after panaxydol treatment (50 g/ml) were analyzed by digital imaging microscopy. bMCF-7 cells were pretreated with DPI (10 M), NAC (5 mM) or apocynin (300  for 1 h and loaded with the Ca2+ indicator, fluo-4AM (green). Changes in [Ca2+]i were analyzed 5 min after panaxydol treatment (50 g/ml) by confocal microscopy. P indicates panaxydol.

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