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 Authors: Cheol-Hee Choi, Yong-Keun Jung and Seon-Hee Oh Article title: Molecular Pharmacology (MOL #63495) SUPPLEMENTARY MATERIAL (Fig.1-3)

Supplementary Fig. 1 MCF10A IRE1 Chop β-actin Caspase-4 17kDa 26 kDa p19 Caspase-7 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.

MCF-7 MDA-MB-231 MCF10A Bcl2 Bid β-actin 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) Supplementary Fig. 2

Fig. 3A Supplementary Fig. 3 p-Akt PD98059 SB Akt Capsaicin DMSO β-actin ERK2 LC3 II p-ERK1/2 Effects of p38 and ERK on capsaicin-induced autophagy in MDA-MB-231 cells. (A) Cells were pretreated with SB (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. Fig. 3B NS siRNA sip38 siRNA control p-Akt Akt β-actin ERK2 LC3 II p-ERK1/2 p-p38 p38 Fig. 3C Capsaicin NS siRNA ERK siRNA control p-Akt T-Akt p-ERK1/2 ERK2 LC3I/ II β-actin