Mitochondrial impairment triggers cytosolic oxidative stress and cell death following proteasome inhibition

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Supplementary Information Mitochondrial impairment triggers cytosolic oxidative stress and cell death following proteasome inhibition Sunita Maharjan, Masahide Oku, Masashi Tsuda, Jun Hoseki 2 & Yasuyoshi Sakai, 2 Division of pplied Life Sciences, Graduate School of griculture, Kyoto University, Kyoto 66-852, Japan, 2 Research Unit for Physiological Chemistry, the Center for the Promotion of Interdisciplinary Education and Research, Kyoto University, Kyoto 66-852, Japan. Figure S Polyubiquitinated proteins accumulate under proteasome inhibition. Immunoblot of polyubiquitinated proteins using anti-ubiquitin antibody. Samples were % NP-4 lysates of CHO cells treated with either DMSO (control) or M bortezomib () for 8 h at 37 C. Figure S2 Resveratrol reduces cytosolic ROS and improves cell viability under proteasome inhibition. () Relative cell viability as determined by the MTT assay. (B) Cytosolic H2O2 detection using HyPer under proteasome inhibition. 2 was used as a positive control. Quantitated values are shown as the means ± s.e.m. of three independent measurements. : p <., : p <.5. Figure S3 Sesamin functions as a redox modulator in cells under proteasome inhibition. () FRET analysis of CHO cells expressing Redoxfluor, (C) cytosolic ROS detection using CM-H2DCFD, (D) mitochondrial membrane potential detection using Mitotracker Red CMXRos, and (E) mitochondrial ROS detection using the MitoSOX Red probe, after treatment with DMSO (control), M bortezomib (), or simultaneous treatment with M bortezomib and M sesamin ( + Ses). Rotenone was used as a positive control for mitochondrial dysfunction and ROS generation. Fluorescence images (left panel) and quantitated fluorescence intensities (right panel) are shown. (B) Relative cell viability as determined by the MTT assay. ll treatments were done for 8 h. Quantitated values are shown as the means ± s.e.m. of three independent measurements. : p <., : p <.5. Figure S4 scorbic acid did not affect cytosolic redox state in cells under proteasome inhibition. FRET images of CHO cells expressing Redoxfluor (upper panel) and quantitated fluorescence intensities (lower panel) are shown. Treatments were done for 8 h. Quantitated values are shown as the means ± s.e.m. of three

independent measurements. : p <., NS: not significant. Figure S5 SOD expression and cellular localization. () nti-mnsod immunoblot of % NP-4 lysates from CHO cells transfected with either MnSOD construct or empty vector (pcdn3.). (B) Mitochondrial localization of overexpressed MnSOD. Immunofluorescence staining of CHO cells transfected with MnSOD. White arrows indicate MnSOD-overexpressing cells. (C) Mitochondrial ROS detection using the MitoSOX Red probe in cells transfected with pcdn3. (mock) after treatment with DMSO (Mock + DMSO), cells transfected with pcdn3. (mock) after treatment with MG32 (Mock + MG32), and cells transfected with MnSOD after treatment with M MG32 (MnSOD + MG32). Under all conditions, cells were transiently transfected with respective plasmids. Fluorescence images (upper panel) and quantitated fluorescence intensities (lower panel) are shown. (D) nti-h immunoblot of % NP-4 lysates from CHO cells transfected with either H-SOD construct or empty vector (pcdn3.). Figure S6 Validation of light mitochondrial fraction and ubiquitin localization under proteasome inhibition. () Validation of the light mitochondrial fractionation under DMSO (control) and M MG32 (MG32) treatment along with whole cell extract using a mitochondrial marker (anti-vdc), a cytosolic marker (anti-gpdh), an endoplasmic reticulum marker (nti-erp72), and a peroxisome marker (nti-catalase) (B) Localization of overexpressed H-Ub. Immunofluorescence staining of CHO cells transfected with H-Ub after treatment with DMSO (H-Ub+DMSO), M ezomib (H-Ub+) and M MG32 (H-Ub+MG32) respectively.

Figure S (kda) 93 6 88 62 47 35 nti-ub 28 2 nti-actin

Figure S2.5 B 2 Relative cell viability MG32 MG32 +Res Relative fluorescence ratio (Ex5/42).5 +Res H 2 O 2

Figure S3 + Ses ezomib 2. (Oxidized) E C ezomib + Ses + Ses + Ses + Ses.25 2.55 2.45 + Ses 2.5.5.25 Rotenone + Ses 2.65.75 Relative fluorescence intensity D.5 Relative fluorescence intensity Relative cell viability B 2.75 Relative fluorescence intensity FRET value 3. (Reduced) FRET ratio 2.85 Rotenone + Ses + Ses

Figure S4 + scorbic acid FRET value 3. (Reduced) 2. (Oxidized) 2.85 2.75 NS FRET ratio 2.65 2.55 2.45 + scorbic acid

M M oc k ns O D Figure S5 B MnSOD Mitotracker Deep Red Merge nti-mnsod nti-actin Mock + MG32 Mock + DMSO MnSOD + MG32 Relative fluorescence intensity C 2 nti-actin D O -S H k oc M nti-h.5 D Mock MnSOD Mock +DMSO +MG32 +MG32

Light Mitochondrial fraction G M C on tr ol 3 ce 2 ll ho m og en at es Figure S6 3kDa nti-vdc 37kDa nti-gpdh 72kDa nti-erp72 65kDa nti-catalase H-Ub +MG32 H-Ub + H-Ub +DMSO B H-Ub Mito tracker Deep Red Merge

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