Authors : Cheol-Hee Choi, Yong-Keun Jung and Seon-Hee Oh

1. Article title: Molecular Pharmacology (MOL #63495) Authors: Cheol-Hee Choi, Yong-Keun Jung and Seon-Hee Oh Journal title: Autophagy Induction by Capsaicin in Malignant Human Breast Cells is Modulated by p38 and ERK Mitogen-Activated Protein Kinases and Retards Cell Death by Suppressing Endoplasmic Reticulum Stress-Mediated Apoptosis SUPPLEMENTARY MATERIAL(Fig.1-3)

2. Supplementary Fig. 1 MCF10A 0 3 6 12 24 IRE1 Chop 26 kDa Caspase-4 p19 17kDa Caspase-7 β-actin Activation of caspase-4 and induction of ER stress-related proteins in MCF-10A cells. Cells were treated with 250 µM capsaicin, harvested at indicated times, lyzed, and the lysates were subjected to immunoblotting. Capsaicin treatment induced ER stress through induction of IRE1, Chop, caspase-4, and caspase-7 activation.

3. 0 0.5 1 3 6 12 24 0 0.5 1 3 6 12 24 0 0.5 1 3 6 12 24 Bcl2 Bid β-actin Supplementary Fig. 2 MCF10A MCF-7 MDA-MB-231 Bcl2 induction responds to capsaicin in malignant breast cells. To examine whether the less sensitivity to capsaicin in malignant breast cells is related with suppression of apoptosis, Expression of Bcl2 and Bid was analyzed Cells were treated with 250 µM capsaicin for up to 24 h, harvested, and lysed, and the Bcl2 and Bid were analyzed by immunoblotting. Antiapoptotic Bcl2 increased in a time-dependent manner in MCF-7 and MDA-MB-231cells. However, Bid remained constant. In MCF10A cells, Bcl2 and Bid downregulated respond to capsaicin (Lee et al., 2009)

4. NS siRNA sip38 siRNA NS siRNA ERK siRNA control DMSO PD98059 SB203580 control - + - + - + Capsaicin - + - + - + - + - + - + Capsaicin p-Akt p-p38 p-Akt p38 Akt T-Akt p-Akt p-ERK1/2 p-ERK1/2 Akt ERK2 ERK2 p-ERK1/2 LC3 II LC3I/ II ERK2 β-actin β-actin LC3 II β-actin Supplementary Fig. 3 Fig. 3B Fig. 3A Fig. 3C Effects of p38 and ERK on capsaicin-induced autophagy in MDA-MB-231 cells. (A) Cells were pretreated with SB203580 (10 μM) or PD98059 (10 μM) for 30 min before 250 µM capsaicin was added for 6 h. Cells were then harvested and lysed, and lysates were analyzed for p38, p-p38, Akt, p-Akt, ERK, p-ERK, and LC3II by immunoblotting. (B) Knockdown of the p38 gene blocked capsaicin-induced autophagy. Cells transfected with nonspecific (NS) siRNA or p38-specific siRNA were treated with capsaicin (250 µM) or DMSO for 6 h, and lysates were analyzed by immunoblotting. (C) Knockdown of the ERK gene enhanced the induction of autophagy. Cells transfected with nonspecific (NS) siRNA or ERK-specific siRNA were treated with capsaicin (250 µM) or DMSO for 6 h, and lysates were analyzed by immunoblotting. The p-Akt antibody used in the present study was not effective in MDA-MB-231 cells. Downregulation of p-p38 blocked LC3 conversion through downregulation of p-Akt. In contrast, downregulation of p-ERK enhanced LC3 conversion downstream of Akt.