Cell - 8 September 2016

Supplemental Figures

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hsp-6 dnj-10 dnj-21 spg-7 phb-2 grpE hsp-90 timm-8 timm-13 timm-17 timm-44

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GFP Fluorescence (RFU)

hsp-16.2p::GFP

hsp-6p::GFP

EV hsp-6 dnj-10 dnj-21 spg-7 phb-2 gfpE RNAi

hsp-90 timm-8 t imm-13 timm-17 timm-44 tomm-20 tomm-40 RNAi

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hsp-16.2p::GFP

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Relative Band IntensityEV hsp-6 tomm-20 EV hsp-6 tomm-20

Cytosolic Mitochondrial

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Chymotrypsin-like proteasome activity

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Figure S1.hsp-6RNAi Induces a MCSR that Is Independent of Mitochondrial Import, Related toFigure 1
(A) qPCR of different compartmental UPR genes afterhsp-6RNAi (mean±SD of three biological repeats).
(B) UPRmtcomponents were required for induction of the cytosolic response.hsp-16.2p::GFP reporter induction was measured by COPAS biosorter (right panel,
mean±SD of three biological repeats). (Continued fromFigure 1C, note that the image of control worms are from theFigure 1C).
(C) RNAi of the other mitochondrial import and quality control genes did not inducehsp-16.2p::GFP reporter (left panel), while they all moderately turned on the
hsp-6p::GFP reporter (right panel, images of GFP induction).hsp-16.2p::GFP induction was measured via ImageExpress software after RNAi treatment (mean±
SEM of three biological repeats).
(D) Mitochondrial HSP60 was detected after the indicated RNAi. The Western blots were quantified by normalizing to NUO-2 expression level (mitochondrial
fraction) or to Tubulin expression level (cytosolic fraction). The band intensity of each sample is compared to the respective EV control. Quantification of relative
band intensity is shown in the lower panel (mean±SD of three biological repeats, *p%0.05, ***p%0.001).
(E) SDS-insoluble proteins after RNAi (Silver staining of SDS-PAGE gel). PBS: PBS-soluble proteins, SDS: SDS-soluble proteins from the PBS-insoluble pellet,
GnHCl: GnHCl extracted proteins from the SDS-insoluble pellet. (F) Proteasome activity. Chymotrypsin-like proteasome activity was measured after indicated
RNAi (Relative slope quantification is shown as a bar graph. mean±SD of two biological repeats).