Supplementary Fig. S1

(1)

0 10 20 30 40 50 60 70 80 90 100

- 25 25 25 50 50 50

- 25 50 100 25 50 100

Cell Death [%]

WT ATG5 KO ATG7 KO

ABT [µM]

ETO [µM]

Supplementary Fig. S1

(2)

0 10 20 30 40 50 60 70 80 90 100

24 48 72

Cell Death [%]

Time [h]

ABT/ETO

WT ATG5 KO ATG7 KO

*

Supplementary Fig. S2

(3)

0 10 20 30 40 50 60 70 80 90 100

UT 10 12,5 15 17,5 20 22,5 25 27,5 30

LN-229 WT LN-229 ATG7 KO

0 10 20 30 40 50 60 70 80 90 100

24 48 72

WT ATG7 KO

0 10 20 30 40 50 60 70 80 90 100

UT 10 12,5 15 17,5 20 22,5

LN-229 WT LN-229 ATG7 KO

0 10 20 30 40 50 60 70 80 90 100

24 48 72

WT ATG7 KO LOP [µM]

PIMO [µM]

0 5 10 15 20 25 30 35 40 45 50

UT 15 20 25 30 35 40

U343 WT U343 ATG5 KO

STF [µM]

0 10 20 30 40 50 60 70 80 90 100

24 48 72

WT ATG5 KO

A B

C D

E F

*

**

*

**

*

***

*

* ** * *

* **

Supplementary Fig. S3

Time [h]

(4)

0 10 20 30 40 50 60 70 80 90 100

UT 50 nM 100 nM 200 nM 400 nM

MZ-54 WT MZ-54 ATG5 KO MZ-54 ATG7 KO

0 10 20 30 40 50 60 70 80 90 100

UT 50 nM 100 nM 200 nM 400 nM

Cell Death [%]

0 10 20 30 40 50 60 70 80 90 100

UT 50 nM 100 nM 200 nM 400 nM

A

C

B

Supplementary Fig. S4

(5)

0 10 20 30 40 50 60 70 80 90 100

UT LOP PIMO STF RSL3

-Trolox +Trolox

0 10 20 30 40 50 60 70 80 90 100

-GSH +GSH

0 10 20 30 40 50 60 70 80 90 100

-a-Toc +a-Toc

0 10 20 30 40 50 60 70 80 90 100

-Trolox +Trolox

0 10 20 30 40 50 60 70 80 90 100

-GSH +GSH

0 10 20 30 40 50 60 70 80 90 100

-a-Toc +a-Toc

Trolox GSH α-TOC

MZ-54 WTMZ-54 ATG7 KO

0 2 4 6 8 10 12 14 16

UT LOP PIMO RSL3

ROS production (foldchange)

WT ATG7 KO

***

**

***

* * ** *

***

** **

**

*** **

A

0 2 4 6 8 10 12 14 16

- - + + - - + + - - + +

UT UT STF STF RSL3 RSL3

ROS production (foldchange)

*

B

Trolox

** * *** *

Supplementary Fig. S5

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IM/TIC LOP

UT 1h 3h 8h 24h 48h

Vinculin LC3B-I LC3B-II

250kDa

Vinculin LC3B-I LC3B-II

130 kDa 100 kDa

2h 6h UT 1h 3h 8h 24h 48h2h 6h

250kDa 130 kDa 100 kDa 15kDa

15kDa

PIMO STF

UT 1h 3h 8h 24h 48h2h 6h UT 1h 3h 8h 24h 48h2h 6h

Supplementary Fig. S6

(7)

15 kDa Vinculin

LC3-I LC3-II

100 kDa 15 kDa

130 kDa

LOP - + - +

25 kDa 55 kDa 70 kDa ATG7

ATG5-ATG12

ATG5

Vinculin LC3-I LC3-II

100 kDa 15 kDa

130 kDa

PIMO - + - +

25 kDa 55 kDa 70 kDa ATG7

ATG5-ATG12

ATG5

Vinculin LC3-I LC3-II

100 kDa 130 kDa

STF - + - +

25 kDa 55 kDa 70 kDa ATG7

ATG5-ATG12

ATG5

Supplementary Fig. S7

LN-229 WT ATG7 KO

U343 GOS-3 WT ATG5 KO

A

C B

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A

B

0 20 40 60 80 100 120

- - + + - - + + - - + + - - + + - - + +

DMSO LOP PIMO STF IM/TIC

Puncta (number/cell)

Yellow puncta Red puncta

*** ***

***

** ***

*

***

*

***

*

BafA1

Supplementary Fig. S8

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1 Supplementary Figure Legends

Supplementary Figure S1. ABT-737 in combination with etoposide induces autophagy-independent cell death in MZ-54 cells.

MZ-54 WT, ATG5 KO and ATG7 KO cells were treated with indicated concentrations of ABT-737/etoposide for 48 hours. Cell death was assessed by measuring the PI uptake as fraction of total nuclei determined by Hoechst counterstaining using high-content fluorescence microscopy. Mean and SEM of three independent experiments performed in triplicate are shown. ABT = ABT-737, ETO = etoposide.

Supplementary Figure S2. ABT-737 in combination with etoposide induces autophagy-independent cell death of MZ-54 in a time-dependent manner.

MZ-54 cells were treated with the indicated concentrations of ABT-737/etoposide for 24, 48 and 72 hours. Cell death was assessed by measuring the PI uptake as fraction of total nuclei determined by Hoechst counterstaining using high-content fluorescence microscopy. Mean and SEM of three independent experiments performed in triplicate are shown. Significances are calculated versus WT cells. * p<0.05. ABT = ABT-737, ETO = etoposide.

Supplementary Figure S3: Loperamide, pimozide and STF-62247 induce autophagy- dependent cell death in several GBM cell lines.

LN-229 WT, LN-229 ATG7 KO, U343 WT and U343 ATG5 KO cells were treated with the indicated concentrations of loperamide (A), pimozide (C) and STF-62247 (E) for 48 hours (A, C) or 72 hours (E) or with 20 µM loperamide (B), 12.5 µM pimozide (D) or 30 µM STF- 62247 (F) for the indicated time points. Cell death was assessed by measuring PI uptake as fraction of total nuclei determined by Hoechst counterstaining using high-content fluorescence microscopy. Data are presented as mean and SEM of three to five

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2 independent experiments performed in triplicate. Significances are calculated against WT cells. * p<0.05, ** p<0.01, *** p<0.001.

Supplementary Figure S4. Rapamycin does not induce cell death in MZ-54 cells.

MZ-54 WT, ATG5 KO and ATG7 KO cells were treated with indicated concentrations of rapamycin for 24 hours (A), 48 hours (B) or 72 hours (C). Cell death was assessed by measuring the PI uptake as fraction of total nuclei determined by Hoechst counterstaining using high-content fluorescence microscopy. Data are presented as mean and SEM of three independent experiments performed in triplicate.

Supplementary Figure S5. Effect of Loperamide, pimozide and STF-62247 on ROS formation.

A MZ-54 WT and ATG7 KO cells were pre-treated with 200 µM trolox, 2.5 mM reduced GSH or 100 µM α-Toc for one hour prior to treatment with 17.5 µM loperamide, 15 µM pimozide, 40 µM STF-62247 or 0.5 µM RSL3 for 48 hours. Cell death was assessed by measuring the PI uptake as fraction of total nuclei determined by Hoechst counterstaining using high-content fluorescence microscopy. Significances of samples treated in the presence of ROS scavengers are calculated against the corresponding samples treated in the absence of ROS scavengers. B MZ-54 WT and ATG7 KO cells were treated with 17.5 µM loperamide, 15 µM pimozide or 40 µM STF-62247 for 24 hours, with 0.5 µM RSL3 for two hours (left panel) or with 0.6 µM RSL3 for 6 hours (right panel). ROS production was determined by FACS analysis of the viable cell population using the fluorescent dye CM- H2DCFDA and is shown as fold change compared with control. Data are presented as mean and SEM of three to five independent experiments performed in triplicate.

Significances are calculated against untreated samples of the corresponding cell line. * p<0.05, ** p<0.01, *** p<0.001. UT = untreated, LOP = loperamide, PIMO = pimozide, STF

= STF-72247.

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3

Supplementary Figure S6: Loperamide, pimozide and STF-62247 induce autophagy of MZ-54 in a time-dependent manner.

MZ-54 cells were treated with 20 µM IM/100 µM TIC, 17.5 µM loperamide, 15 µM pimozide and 40 µM STF-62247 for the indicated time points followed by detection of LC3B and vinculin protein levels by Western blotting with vinculin as loading control. UT = untreated, IM = imipramine hydrochloride, TIC = ticlopidine, LOP = loperamide, PIMO = pimozide, STF = STF-62247.

Supplementary Figure S7: Loperamide, pimozide and STF-62247 induce autophagy in LN-229 and U343 GOS-3 cells.

LN-229 WT and ATG7 KO cells as well as U343 WT and ATG5 KO cells were treated with 20 µM loperamide (A), 12.5 µM pimozide (B) and 30 µM STF-62247 (C) for 24 hours followed by detection of ATG7, ATG5, LC3B and vinculin protein levels by Western blotting with vinculin as loading control. UT = untreated, LOP = loperamide, PIMO = pimozide, STF = STF-62247.

Supplementary Figure S8. Loperamide, pimozide and STF-62247 enhance the autophagic flux of MZ-54 cells.

A MZ-54 cells stably expressing mRFP-GFP-LC3B were treated with 20 µM IM/100 µM TIC, 15 µM loperamide, 15 µM pimozide and 40 µM STF-62247 for 30 hours in the absence or presence of 40 nM BafA1 followed by fluorescence microscopy of mRFP-GFP- LC3B and DAPI-stained nuclei. Scale bar = 30 µm. B Quantification of yellow and red puncta was performed for three independent experiments with three to five images per sample. Significances are calculated against the corresponding DMSO sample in the presence or absence of BafA1. * p<0.05, ** p<0.01, *** p<0.001. LOP = loperamide, PIMO

= pimozide, STF = STF-62247, IM = imipramine hydrochloride, TIC = ticlopidine.