Mitochondrial impairment triggers cytosolic oxidative stress and cell death following proteasome inhibition
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1 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 , Japan, 2 Research Unit for Physiological Chemistry, the Center for the Promotion of Interdisciplinary Education and Research, Kyoto University, Kyoto , 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
2 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.
3 Figure S (kda) nti-ub 28 2 nti-actin
4 Figure S2.5 B 2 Relative cell viability MG32 MG32 +Res Relative fluorescence ratio (Ex5/42).5 +Res H 2 O 2
5 Figure S3 + Ses ezomib 2. (Oxidized) E C ezomib + Ses + Ses + Ses + Ses Ses Rotenone + Ses 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
6 Figure S4 + scorbic acid FRET value 3. (Reduced) 2. (Oxidized) NS FRET ratio scorbic acid
7 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
8 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
Supplementary Materials
Supplementary Materials Figure S1. MTT Cell viability assay. To measure the cytotoxic potential of the oxidative treatment, the MTT [3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide] assay
293T cells were transfected with indicated expression vectors and the whole-cell extracts were subjected
SUPPLEMENTARY INFORMATION Supplementary Figure 1. Formation of a complex between Slo1 and CRL4A CRBN E3 ligase. (a) HEK 293T cells were transfected with indicated expression vectors and the whole-cell
Additional methods appearing in the supplement are described in the Experimental Procedures section of the manuscript.
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
Appendix. Table of Contents
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DOI:.38/ncb2822 a MTC02 FAO cells EEA1 b +/+ MEFs /DAPI -/- MEFs /DAPI -/- MEFs //DAPI c HEK 293 cells WCE N M C P AKT TBC1D7 Lamin A/C EEA1 VDAC d HeLa cells WCE N M C P AKT Lamin A/C EEA1 VDAC Figure
Supplementary figure legends
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Type of file: PDF Title of file for HTML: Supplementary Information Description: Supplementary Figures Type of file: MOV Title of file for HTML: Supplementary Movie 1 Description: NLRP3 is moving along
hexahistidine tagged GRP78 devoid of the KDEL motif (GRP78-His) on SDS-PAGE. This
SUPPLEMENTAL FIGURE LEGEND Fig. S1. Generation and characterization of. (A) Coomassie staining of soluble hexahistidine tagged GRP78 devoid of the KDEL motif (GRP78-His) on SDS-PAGE. This protein was expressed
Supplementary Figure 1: GFAP positive nerves in patients with adenocarcinoma of
SUPPLEMENTARY FIGURES AND MOVIE LEGENDS Supplementary Figure 1: GFAP positive nerves in patients with adenocarcinoma of the pancreas. (A) Images of nerves stained for GFAP (green), S100 (red) and DAPI
(A) SW480, DLD1, RKO and HCT116 cells were treated with DMSO or XAV939 (5 µm)
Supplementary Figure Legends Figure S1. Tankyrase inhibition suppresses cell proliferation in an axin/β-catenin independent manner. (A) SW480, DLD1, RKO and HCT116 cells were treated with DMSO or XAV939
SUPPLEMENTARY INFORMATION
DOI: 10.1038/ncb2566 Figure S1 CDKL5 protein expression pattern and localization in mouse brain. (a) Multiple-tissue western blot from a postnatal day (P) 21 mouse probed with an antibody against CDKL5.
Supplementary Figure 1
Supplementary Figure 1 14 12 SEM4C PLXN2 8 SEM4C C 3 Cancer Cell Non Cancer Cell Expression 1 8 6 6 4 log2 ratio Expression 2 1 4 2 2 p value.1 D Supplementary Figure 1. Expression of Sema4C and Plexin2
Supplementary Figure 1
Supplementary Figure 1 Supplementary Figure 1 SNARE Probes for FRET/2pFLIM Analysis Used in the Present Study. mturquoise (mtq) and Venus (Ven) are in blue and yellow, respectively. The soluble N-ethylmaleimide-sensitive
Supplementary Figure 1. MAT IIα is Acetylated at Lysine 81.
IP: Flag a Mascot PTM Modified Mass Error Position Gene Names Score Score Sequence m/z [ppm] 81 MAT2A;AMS2;MATA2 35.6 137.28 _AAVDYQK(ac)VVR_ 595.83-2.28 b Pre-immu After-immu Flag- WT K81R WT K81R / Flag