of 12
Molecular Cell, Volume
84
Supplemental information
The HRI branch of the integrated stress response
selectively triggers mitophagy
Yogaditya Chakrabarty, Zheng Yang, Hsiuchen Chen, and David C. Chan
Figure S1. Quantification of DFP
-
induced mitophagy. Related to Figure 1.
(
A
) Schematic
showing how
mito
-
mKeima
functions
as a fluorescent mitophagy marker. Mito
-
mKeima
has excitation properties that are pH sensitive. By tracking the acidic/neutral ratio of
mKeima, mitochondria within lysosomes
can
be distinguished from normal mitochondria. (
B
)
Dose
-
dependence of DFP
-
induced mitophagy. K562 cells were treated with the indicated
concentrations of DFP for 24 h, and mitophagy was quantified by flow cytometry as in Figure 1B
(mean ± s.d.,
n
≥3).
The following
p
-
value designations are used in all figures: ****, p≤0.0001; ***,
p≤0.001; **, p≤0.01; *, p≤0.05; ns, p≥0.05.
(
C
) Inhibition of DFP
-
induced mitophagy by addition of
haemin (mean ± s.d.,
n
=2). (
D
) Representati
ve flow cytometry plots for K562 cells expressing
mito
-
mKeima and either control sgRNA (sgNT) or sgRNA against HRI. The
pol
ygon
indicates the
gate used to score cells as positive for mitophagy. (
E
) Effectiveness of sgRNA knockdowns. K562
cells expressing the indicated sgRNA were treated with DFP and analyzed by immunoblotting.
NT, non
-
targeting. (
F
) Effectiveness of sgRNA
-
DELE1 knockdown. Quantitative polymerase
chain reaction (qPCR) was used to quantify DELE1 mRNA levels in K562 cells expressing non
-
target
ing (NT) sgRNA or sgRNA against DELE1 (mean ± s.d.,
n
=3). (
G
) Time course of TIMM50
in DFP
-
treated K562 cells. Cell lysates were analyzed by immunoblotting against TIMM50. (
H
)
Immunoblot analysis of TIMM50 in K562 cells expressing the indicated sgRNA, after 23 h DFP
treatment. (
I
) Volcano plot of CRISPRi screen, showing select genes of interest.
Figure S2. HRI components required for DFP
-
induced mitophagy. Related to Figure 2.
(
A
) Inhibition of ATF4 upregulation by DELE1 and HRI knockdowns. K562 cells expressing non
-
targeting (NT) sgRNA or sgRNAs against DELE1 and HRI were treated with DFP and analyzed
by immunoblotting for ATF4. (
B
) Effectiveness of ATF4 sgRNA knockdown by immunoblot
analysis. DFP treatment was used to induce ATF4 expression, so that ATF4 knockdown efficiency
could be evaluated. (
C
) Effectiveness of shRNA
-
HRI and shRNA
-
OMA1 knockdown by
immunoblot analysis. (
D
) Effectiveness of shRNA
-
DELE1 knockdown by qPCR ana
lysis (mean ±
s.d.,
n
=3).
The following
p
-
value designations are used in all figures: ****, p≤0.0001; ***, p≤0.001;
**, p≤0.01; *, p≤0.05; ns, p≥0.05.
(
E
) Effectiveness of shRNA
-
ATF4 knockdowns by immunoblot
analysis. (
F
) Effect of knockdown of HRI components on DFP
-
induced mitophagy in HeLa cells.
Mitophagy was quantified by flow cytometry (mean ± s.d.,
n
≥3). DFP was used at 1 mM. (
G
) Effect
of ATF4 knockdown on DFP
-
induced mitophagy in HeLa cells, at both 250
μ
M (top panel) and 1
M (bottom panel). (
H
) Effect of CHX on mitophagy. K562 cells expressing mito
-
mKeima were
treated with different concentrations of CHX, and flow cytometry was used to measure mitophagy.
DFP treatment was used as a positive control (mean ± s.d.,
n
=2). (
I
) Effect of translation inhibitors
on protein puromycylation. After treatment of cells with the indicated compounds, puromycin was
applied to release translating polypeptides, and cell lysates were analyzed by Western blotting
with an anti
-
puromycin antibody.
Figure S3. Effect of the ISR on mitophagy. Related to Figure 3.
(
A
) Immunoblotting of GFP (control) and CR
e
P
-
GFP in K562 cells. (
B
) Mitophagy induction by
drugs that activate the HRI pathway. Btz, bortezomib. (
C
) Induction of p
-
EIF2
a
by the drugs in
(
B
). (
D
) Accumulation of ATF4 and LC3B lipidation by ISR
-
inducing drugs. (
E
) Mitophagy levels
in
K562
cells expressing the indicated sgRNA, after treatment with ISR
-
inducing drugs.
The
control
/untreated
cells expressing sgNT
were used to set the baseline level of mitophagy.
(
F
)
Effect of ISRIB treatment on mitophagy levels, with and without DFP.
The following
p
-
value
designations are used in all figures: ****, p≤0.0001; ***, p≤0.001; **, p≤0.01; *, p≤0.05; ns, p≥0.05.
Figure S4. Subcellular localization of p
-
EIF2α after BTd and Sal treatment. Related to
Figure 4.
(
A
) Comparison of endoplasmic reticulum (Sec61b
-
Emerald) and mitochondrial (mito
-
DsRed2)
signals in HeLa cells. (
B
) Biochemical localization of p
-
EIF2
a
to mitochondria after DFP treatment
of HeLa cells. Samples were processed as in Figure 4A. TOM20, mitochondrial marker. (
C
)
Comparison of p
-
EIF2
a
levels in different cellular fractions of Hela cells treated with DFP.
Endoplasmic reticulum (ER) enriched fractions were obtained by ultracentrifugation of post
-
mitochondrial supernatant at 100,000
g
. Post
-
ER supernatant was used as the
cytosolic (Cyto
-
II)
fraction. CNX (Calnexin) and VDAC1 were used as ER and mitochondrial markers, respectively.
(
D
) Immunolocalization of p
-
EIF2
a
in HeLa cells after BTd and Sal treatment. Cells were treated
with 10 mM BTd or 10 mM Sal for 24 h and analyzed by immunofluorescence. Inset
s
show
magnified image of boxed area.