SUPPLEMENTARY DATA

Interplay between TRAP1 and sirtuin-3 (SIRT3) modulates mitochondrial respiration and oxidative stress to maintain glioma stem cells

Hye-Kyung Park1,6, Jun-Hee Hong2,6, Young Taek Oh2, Sung Soo Kim2, Jinlong Yin2, An-Jung Lee1, Young Chan Chae1, Jong Heon Kim2, Sung Hye Park3, Chul-Kee Park3, Myung-Jin Park4, Jong Bae Park2,5,*, and Byoung Heon Kang1,*

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Contents

Supplementary Tables ……….………………………………………………………………………. 3

Supplementary Figures

Supplementary Figure S1. Expression of TRAP1 and Sox2 ……………………………... 4

Supplementary Figure S2. Effect of TRAP1 on GSC metabolism ………………………. 6

Supplementary Figure S3. Mitochondrial respiration and ROS production in glioma

stem cells ………………………………………………………

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Supplementary Figure S4. Sphere formation after TRAP1 activation………….…...…

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Supplementary Figure S5. TRAP1 chaperones SIRT3, which regulates mitochondrial

ROS production by, and viability of, GSCs ………..………

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Supplementary Figure S6. Regulation of mitochondrial respiration…….………….… 14

Supplementary Figure S7. Sphere and colony formation after SIRT3 knockdown..… 15

Supplementary Figure S8. TRAP1 inhibition reduces the tumor-forming activity of

GSCs

in vivo ……………...………………………………………… 16

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Supplementary Tables

Table S1. Immunohistochemical analysis of TRAP1 expression in brain tumor specimens

TRAP1 expression Neg. 1+ 2+ 3+ Total cases

Grade 2 5 7 3 0 15

Grade 3 4 24 10 1 39

Grade 4 3 34 45 14 96

Total cases 12 65 58 15 150

Table S2. Immunohistochemical analysis of Sox2 expression in brain tumor specimens

Sox2 expression Neg. 1+ 2+ 3+ Total cases

Grade 2 2 8 4 1 15

Grade 3 3 17 14 5 39

Grade 4 4 30 44 18 96

Total cases 9 55 62 24 150

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Supplementary Figure S1. Expression of TRAP1 and Sox2.

A, Scatter plots showing expression of TRAP1 and Sox2. LN229 and cancer stem cell (CSC)-

enriched LN229 cells were fixed, permeabilized, and stained with rabbit anti-TRAP1 or goat anti-

Sox2 antibodies, followed by Alexa Fluor 488-conjugated anti-rabbit IgG or Alexa Fluor 633-

conjugated anti-goat IgG antibodies, prior to analysis by flow cytometry. B, Scatter plot showing

expression of TRAP1 and Sox2 in glioma stem cells (GSCs). X01 cells were stained and analyzed as

in (A). Pearson’s correlation coefficient (r) was calculated to determine the correlation between

expression of TRAP1 and Sox2 in (A) and (B). C, D, Overall survival of all glioma patients showing

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differential expression of TRAP1 or Sox2. Kaplan-Meier survival curves were generated from data

derived from all glioma patient specimens investigated in this study. E, Survival of glioma patients

showing high expression of both TRAP1 and Sox2. Kaplan-Meier plots were generated for patients

showing high and low expression of both TRAP1 and Sox2. (C–E) Scores of ≥ 2+ and ≤ +1 denote

high and low expression, respectively. The total number of patients with high and low expression is

indicated. P values were calculated using the log-rank test. F, G, Correlation between Sox2 and

TRAP1 expression in glioma patients. Staining scores for TRAP1 and Sox2 in all glioma (F) or

glioblastoma patients (G) were used to calculate Spearman’s correlation coefficient (rs).

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Supplementary Figure S2. Effect of TRAP1 on metabolism of GSC.

A, Mitochondrial membrane potential. TMRM-labeled X01 cells were incubated for 12 h with 1 μM

gamitrinib and analyzed by flow cytometry. B, Lactate production. X01 cells were treated for 12 h

with gamitrinib (as indicated), and relative lactate concentrations in the medium were analyzed. **,

p=0.006. C, TRAP1 expression by enriched cancer stem cells (CSCs). CSCs (serum -) were enriched

from GBM cell lines (serum+) as described in Materials and methods. Whole cell lysates were

analyzed by western blotting with appropriate antibodies. D, Measurement of the oxygen

consumption rate (OCR). CSC-enriched ((S)LN229) and parental LN229 cells were incubated for 24

h with 0.5 μM gamitrinib (Gami) and data were analyzed using the XF analyzer. E, Lactate

production. (S)LN229 cells were incubated with 2 μM gamitrinib (Gami) for 12 h and the lactate

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concentration in the medium was measured. **, p=0.008. F, Mitochondrial production of reactive

oxygen species. Mito-SOX-labeled (S)LN229 cells were incubated for 6 h with 1 μM gamitrinib and

then analyzed by flow cytometry.

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Supplementary Figure S3. Mitochondrial respiration and ROS production by glioma stem cells.

A, Differentiation of X01 cells. X01 cells were cultured in the presence (+, differentiated) or absence

(-, maintaining stemness) of serum and analyzed by western blotting. B, Glucose uptake. Glucose

uptake by X01 cells cultured in the presence or absence of serum was measured and compared. *,

p=0.015. C, Lactate production. Lactate concentrations in fresh culture medium were measured after

3 h. ***, p=0.0004. D, Cellular ATP concentration. ATP concentrations were measured and compared.

**, p=0.0059. E, Measurement of the OCR. X01 cells maintained as indicated were analyzed using

the Seahorse XF analyzer. O, F, R, and A indicate treatment with oligomycin, FCCP, rotenone, and

antimycin A, respectively. F, ROS production. X01 cells maintained in medium with or without serum

were labeled with Mito-SOX and analyzed by flow cytometry. G, Analyses of stem cell marker

expression. X01 cells were treated with antimycin A and oligomycin for 2 days, or with H 2O2 for 7

days (as indicated) and analyzed by western blotting. H, Effect of antimycin A on sphere formation.

X01 cells were incubated with antimycin A as indicated and analyzed in a limiting dilution assay

(left). Representative microscopic images (μM, right). I, Oligomycin treatment. X01 cells were

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incubated with oligomycin as indicated and analyzed in a limiting dilution assay. Representative

microscopic images (μM, right). J, Effect of ROS on sphere formation. X01 cells were incubated with

H2O2 as indicated and analyzed in a limiting dilution assay. Representative microscopic images (μM,

right). (B)–(D) Data are expressed as the mean ± SEM of duplicates (at least) in two independent

experiments.

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Supplementary Figure S4. Sphere formation after TRAP1 activation.

A, Gamitrinib treatment. X01 cells were incubated with 0.5 μM gamitrinib for the indicated times and

analyzed under a microscope (left). The number of spheres >100 µm in diameter was counted (right).

Scale bar, 100 µm. B, Sphere formation after treatment with TRAP1 siRNA. X01 cells were treated

with TRAP1 siRNAs and analyzed under a microscope on Day 5 (left). The number of spheres >100

µm in diameter was counted (middle). TRAP1 knockdown efficiency (right). **p<0.002. Scale bar,

100 μm. C, Colony formation after TRAP1 knockdown. X01 cells were treated with TRAP1 siRNAs

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and cultured in cancer stem cell medium for 7 days. Cells were treated for 12 h with 10% FBS to

allow attachment. Next, cells were stained with crystal violet (left) and the number of colonies was

counted (right).

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Supplementary Figure S5. TRAP1 chaperones SIRT3, which regulates mitochondrial ROS

production by, and viability of, GSCs.

A, Cycloheximide treatment. X01 and 528NS cells were incubated with 20 µM cycloheximide (CHX)

in the presence or absence of 1 μM gamitrinib for the indicated times and then analyzed by western

blotting. B, Normalization of SIRT3 enzyme activity to the protein level. SIRT3 enzyme activity

(measured in Fig. 4G) was normalized to the SIRT3 protein level (left panel) from western blot

analysis (right panel). ***, p<0.0001. C, SIRT3 enzyme activity after short-term inhibition of TRAP1.

Mitochondrial extracts from X01 cells were incubated with drugs for 30 min (as indicated) and SIRT3

enzyme activity was analyzed (top). Western blot analysis of whole cell extracts (bottom). Data are

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expressed as the mean ± SEM of two independent experiments performed in duplicate. ***,

p<0.0002. D, Mitochondrial ROS production. SIRT3 siRNA-treated X01 cells were labeled with

Mito-SOX and analyzed by flow cytometry (left) and western blotting (right). E, Overexpression of

SIRT3. SIRT3-overexpressing X01 cells were incubated for 24 h with 1 μM gamitrinib and analyzed

in an MTT assay (top) and by western blotting (bottom). Data are expressed as the mean ± SEM of

two independent experiments performed in duplicate. **, p<0.01. F, Acetylation of mitochondrial

proteins. X01 cells were incubated for 12 h with 5 μM gamitrinib and analyzed by western blotting.

G, Acetylation of SOD2. Mitochondrial extracts from TRAP1 siRNA-treated X01 cells were

immunoprecipitated with an anti-SOD2 antibody and analyzed by western blotting (upper panel).

TRAP1 knockdown efficiency (lower panel). H, SOD2 protein expression. After X01 cells were

incubated for 48 h with 0.5 μM gamitrinib, mitochondrial fractions were obtained and analyzed by

western blotting.

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Supplementary Figure S6. Regulation of mitochondrial respiration.

A, Overexpression of SIRT3. X01 cells transfected with pcDNA3 or pcDNA3-SIRT3 were treated for

24 h with 0.5 μM gamitrinib, and analyzed using an XF analyzer. *p=0.0184; **p=0.0067; NS, not

significant. B, Western blot analysis after SIRT3 overexpression. X01 cells transfected with pcDNA3

or pcDNA3-SIRT3 were treated for 24 h with 0.5 μM gamitrinib, and analyzed by western blotting. C,

Blue native PAGE analysis. Mitochondria were isolated from X01 cells treated for 48 h with DMSO

or 0.5 μM gamitrinib, and analyzed by blue native PAGE and western blotting. Complexes I, II, and V

were detected by antibodies specific for NDUFA9, SDHB, and ATPB, respectively.

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Supplementary Figure S7. Sphere and colony formation after SIRT3 knockdown.

A, Sphere formation. X01 cells were treated with SIRT3 siRNAs and analyzed by phase contrast

microscopy (left). The number of spheres > 100 μm in diameter was counted (right). B, Colony

formation. X01 cells were treated with SIRT3 siRNAs and analyzed in a colony forming assay.

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Supplementary Figure S8. Inhibition of TRAP1 reduces the tumor-forming activity of GSCs in

vivo.

A, TRAP1 knockdown. shControl- or shTRAP1 lentivirus-infected X01 cells (1×104) were

orthotopically implanted into the brains of nude mice. After 47 days, the mice were euthanized and

brain sections were prepared and stained with H&E. B, Tumor growth. 528NS (left) and X01 GSCs

(right) (1×106 cells) were implanted subcutaneously into nude mice. After tumors were established,

mice were injected intraperitoneally with vehicle or 10 mg/kg gamitrinib (daily for 24 days (528NS)

or for 12 days (X01)). Tumors were then collected and photographed. C, Western blot analyses.

Collected tumors (from four mice per group) were lysed and analyzed by western blotting. D,

Quantitation of protein expression. The intensity of protein bands derived from (C) was measured.

Data were normalized to those from β-actin. *, p<0.05. #, not significant.

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