Additional methods appearing in the supplement are described in the Experimental Procedures section of the manuscript.

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Kathryn Holt
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1 Supplemental Materials: I. Supplemental Methods II. Supplemental Figure Legends III. Supplemental Figures Supplemental Methods Cell Culture and Transfections for Wild Type and JNK1-/-,JNK2-/- MEFs: The wild type (WT) and JNKnull MEFs were a kind gift of Dr. Roger Davis (HHMI; University of Massachusetts). MEFs were cultured in as described in the Experimental Procedures. The plasmid DNA for the pcdna3:flag:jnk1α1 was purchased from Addgene, Inc. Cells were grown to ~50% confluency prior to transfection with the JNK1α1 expression plasmid using Fugene6 (Roche) at a 3:2 reagent to DNA ratio according to manufacturer instructions. Following 72 hours, cells were examined for protein expression by western blotting for FLAG and JNK. Western blotting was performed as described in the Experimental Procedures. The antibody for the FLAG epitope was purchased from Cell Signaling Technologies (#2368). Cell Viability: Cellular Viability was monitored by Cayman Chemical s MTT Cell Proliferation Assay Kit Protocol ( ) in accordance with manufacturer s instructions. Given the reliance of the MTT assay on functioning mitochondria, we validated the viability results using SYTOX-Green (Invitrogen) staining and Trypan blue (BioRAD) exclusion counting on the BioRAD TC10 cell analyzer. Additional methods appearing in the supplement are described in the Experimental Procedures section of the manuscript. Supplemental Figure Legends Supplemental Figure S1: JNK selective inhibitor, SR-3562, inhibits ROS generation in a dose dependent manner in anisomycin-stressed HeLa cells. HeLa cells were pretreated with SR-3562 ( nM) for 30 minutes, and then cells were treated with 25mM anisomycin for 35minutes prior to staining with DHE. Oxidized DHE fluorescence was monitored at 45 minutes post-anisomycin introduction. Error bars represent standard deviation from the mean. Supplemental Figure S2: JNK-null MEFs do not display sensitivity to anisomycin-stress, nor do JNK-null MEFs have increased ROS generation or decreased oxygen consumption during stress. (A) JNK-null MEFs do not have decrease cell viability following anisomycin stress. WT and JNK-null cells were treated for 4 hours with 25μM anisomycin, and cell viability was monitored by MTT proliferation assay. (B) JNK-null MEFs do not display increased ROS generation in response to anisomycin stress. WT and JNK-null MEFs were treated with 25μM anisomycin for 35 minutes followed by DHE staining for cellular ROS. ROS-induced fluorescence was monitored at 45 minutes of anisomycin stress. (C) JNK-null MEFs do not generated mitochondrial superoxide in response to anisomycin. WT and JNK-null MEFs were stressed with 25μM anisomycin for 45 minutes, and then cells were stained with MitoSOX-Red for mitochondrial superoxide detection. (D) JNK-null MEFs do not have a decrease in oxygen consumption as a result of anisomycin stress. WT and JNK-null MEFs were stressed with 25μM anisomycin for 40 minutes. Cells were then monitored for oxygen consumption using the Seahorse Biosciences XF-96 Flux Analyzer. Supplemental Figure S3: Complementation of JNK-null MEFs with JNK1α1 results in ROS generation and decreased viability during anisomycin stress. (A) JNK-null MEFs were either mock or transiently transfected with pcdna3:flag:jnk1α1 followed by western blot analysis for the FLAG epitope, JNK, and α-tubulin was used as a loading control. (B) Transient expression of JNK1α1 for 72 1

2 hours was followed by 35 minutes of stress with 25μM anisomycin. WT, JNK-Null and JNK-nulll cells expression JNK1α1 were stained with DHE, and then fluorescence was monitored at 45 minutes post- anisomycin addition. Supplemental Figure S4: Silencing Sab and JNK prevents anisomycin-induced celll death. Cells were transfected with either 50nM control, 50nM Sab-specific, or 50nM JNK-specific sirnas. After 72 hours of silencing, cells weree treated with 25mM anisomcyin for 4 hours, and then cell viability was determined by a MTTT proliferation assay. Supplemental Figures Supplemental Figure S1 2

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SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION Figure S1 Induction of non-apoptotic death of SV40-transformed and primary DKO MEFs, and DKO thymocytes. (A-F) STS-induced non-apoptotic death of DKO MEF. (A, B) Reduced viability of DKO MEFs after exposure