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  • Supplementary figures

    Overview:

    Fig. S1 Overview of significantly altered metabolites determined

    during untargeted analysis with the accurate-mass Q-TOF

    LC-MS system – p 1

    Fig. S2 Example-peaks of metabolites affected by MPP+ treatment

    – p 2

    Fig. S3 MPP+-induced changes of amount and location of

    apoptosis-related proteins – p 3

    Fig. S4 Time course of MPP+ induced ROS generation – p 4

    Fig. S5 Principal component analysis (PCA) of regulated genes of

    MPP+ treated LUHMES cells – p 5

    Fig. S6 Regulation of proteins involved in cellular thiol supply by

    MPP+ – p 6

    Fig. S7 Transcriptional changes triggered by the complex I

    inhibitor rotenone in LUHMES cells – p 7

    Fig. S8 Integrated analysis of metabolomics and transcriptomics

    data to identify affected pathways – p 8

    Fig. S9 Overview of MPP+-induced adaptations in human

    dopaminergic neurons – p 9

    Fig. S10 Antibodies – p 10

    Fig. S11 Primers – p 11

    Transcriptional and metabolic adaptation of

    neurons to the mitochondrial toxicant MPP+

    Anne K. Krug1, Simon Gutbier1, Liang Zhao2, Dominik Pöltl1,3, Cornelius

    Kullmann1, Violeta Ivanova3,4, Sunniva Förster1, Smita Jagtap5, Johannes

    Meiser6, Gérman Leparc7, Stefan Schildknecht1, Martina Adam1, Karsten

    Hiller6, Hesso Farhan8, Thomas Brunner9, Thomas Hartung2, Agapios

    Sachinidis5, Marcel Leist1

  • Downregulated metabolites

    A TP

    C yc

    lic A

    D P -r ib

    os e

    D ec

    an oi

    c ac

    id

    D eh

    yd ro

    as co

    rb at

    e

    D eo

    xy ur

    id in

    e

    D -E

    ry th

    ro se

    D -G

    lu co

    se

    G lu

    ta th

    io ne

    G ua

    ni ne

    In os

    in e

    L- A la

    ni ne

    L- A sp

    ar ag

    in e

    L- G lu

    ta m

    at e

    L- P ro

    lin e

    L- S er

    in e

    M al

    ea m

    at e

    N -A

    ce ty

    l-L -a

    sp ar

    ta te

    N -A

    ce ty

    l-L -g

    lu ta

    m at

    e

    O -A

    ce ty

    l-L -h

    om os

    er in

    e

    O -A

    ce ty

    ln eu

    ra m

    in ic

    a ci

    d

    P an

    to th

    en at

    e

    P ho

    sp ho

    cr ea

    tin e

    sn -g

    ly ce

    ro -3

    -P ho

    sp ho

    et ha

    no la

    m in

    e

    S or

    bi to

    l

    Ta ur

    in e

    U D P -a

    lp ha

    -D -g

    al ac

    to se

    U D P -g

    lu co

    se

    U D P -N

    -a ce

    ty l-D

    -g al

    ac to

    sa m

    in e

    U D P -N

    -a ce

    ty l-D

    -G lu

    co sa

    m in

    e

    2, 3-

    D im

    et hy

    lm al

    ea te

    2, 5-

    D io

    xo pe

    nt an

    oa te

    3- O xo

    pr op

    an oa

    te

    4- A m

    in ob

    ut an

    oa te

    0

    50

    100

    150

    N o

    rm a li z e d

    I n

    te n

    s it

    y V

    a lu

    e s

    [% o

    f c o

    n tr

    o l 

    S D

    ]

    Upregulated metabolites

    A de

    ni ne

    A D P

    A M

    P

    C ho

    le st

    er ol

    s ul

    fa te

    C re

    at in

    e

    D eo

    xy ri bo

    se

    D ih

    yd ro

    pt er

    oa te

    Fo rm

    yl -N

    -a ce

    ty l-5

    -m et

    ho xy

    ky nu

    re na

    m in

    e

    L- A rg

    in in

    e

    L- C ys

    ta th

    io ni

    ne

    L- La

    ct at

    e

    L- Ly

    si ne

    L- M

    et hi

    on in

    e S -o

    xi de

    L- P he

    ny la

    la ni

    ne

    L- Tr

    yp to

    ph an

    L- Ty

    ro si

    ne

    P yr

    uv at

    e

    S -A

    de no

    sy l-L

    -h om

    oc ys

    te in

    e

    S -A

    de no

    sy l-L

    -m et

    hi on

    in e

    S -M

    et hy

    l G S H

    Th ia

    m in

    e ac

    et ic

    a ci

    d

    U ra

    te -3

    -r ib

    on uc

    le os

    id e

    2- A ce

    to la

    ct at

    e

    3- (4

    -H yd

    ro xy

    ph en

    yl )la

    ct at

    e

    3- M

    et hy

    l-2 -o

    xo bu

    ta no

    ic a

    ci d

    4- H yd

    ro xy

    -4 -m

    et hy

    lg lu

    ta m

    at e

    0

    100

    200

    300

    400

    2000

    4000

    control

    24 h

    36 h

    N o

    rm a li z e d

    I n

    te n

    s it

    y V

    a lu

    e s

    [% o

    f c

    o n

    tr o

    l 

    S D

    ]

    B

    A Significantly changed metabolites

    Page 1

    Figure S1

    Overview of significantly altered metabolites determined during untargeted

    analysis with the accurate-mass Q-TOF LC-MS system Cells were treated with 5 µM MPP+ (control, 24 h or 36 h) in four independent experiments.

    After metabolite extraction, samples were run with the accurate-mass Q-TOF LC-MS system

    (Agilent, Santa Clara, CA). Metabolites were determined by the MassHunter Acquisition

    software (Agilent Technologies). ‘Areas under the Curve’ (AUC) for every peak/metabolite of

    the ion chromatogram were calculated and used as basis for a relative quantification. AUCs

    were normalized to the mean of the AUCs of control samples (untreated cells) of

    4 independent experiments. Only metabolites that were significantly regulated (FDR adjusted

    p-value of ≤ 0.05) and that could be unambiguously identified, are displayed.

  • Creatine

    Counts vs. Aquisition Time

    Phosphocreatine

    Counts vs. Aquisition Time

    L-methionine-S-oxide

    Counts vs. Aquisition Time

    S-Adenosyl-L-methionine

    Counts vs. Aquisition Time

    Example-peaks of significantly changed metabolites

    Control

    MPP+

    Page 2

    Figure S2

    Example-peaks of metabolites affected by MPP+ treatment Cells were treated with MPP+ or solvent for 24 h. Samples were run with the accurate-mass

    Q-TOF LC-MS system. Peak overlays are displayed by the Agilent MassHunter Acquisition

    software. The areas under the curve (integral of the peak curve) were used for quantification.

    Data are examples of typical primary data in a representative experiment.

  • Marker expression

    0 12 24 36 48

    0

    50

    100

    Time [h]

    B c l- xL

    [ %

    o f

    c o n tr

    o l  S

    D ]

    [10 h,

    200 nM]

    STS w/o 0 12 24 36 48

    Cytosolic

    cytochrome c

    5 µM MPP+

    GAPDH

    Bcl-xL

    GAPDH

    0 6 12 24 36 48

    5 µM MPP+

    PSPC1

    Tuji

    0 6 12 24 36 48

    5 µM MPP+

    0 12 24 36 48

    0

    10

    20

    30

    40

    Time [h]

    C y to

    s o lic

    c y to

    c h ro

    m e

    c

    [% o

    f p o s it iv

    e c

    o n tr

    o l  S

    D ]

    0 12 24 36 48

    0

    50

    100

    Time [h]

    P S

    P C

    1

    [ %

    o f

    c o n tr

    o l  S

    D ]

    B

    A

    C

    Representative blots: Densitometric quantifications:

    Time [h]:

    Time [h]:

    Time [h]:

    Page 3

    *

    *

    *

    * *

    Figure S3

    MPP+-induced changes of amount and location of apoptosis and cell stress-

    related proteins LUHMES cell lysates were prepared at different times after exposure to MPP+ as illustrated in

    Fig. 1A. Proteins were quantified by western blot. To the left, representative blots are shown.

    To the right, densitometric quantifications of the respective proteins are displayed as means

    ± SD of 3 independent differentiations. Calibration was relative to loading control and

    untreated cell samples. A) Bcl-xl levels. B) The cytosolic cytochrome c levels were determined

    by extraction of soluble cytosolic proteins after permeabilisation of the outer cell membrane

    with 50 µg/ml digitonin. This procedure did not permeabilize the outer mitochondrial

    membrane (Single et al. 1998). Controls were healthy cells without digitonin (w/o), healthy

    cells with digitonin (0) and a positive control of cells exposed to 200 nM staurosporine for 10 h

    (STS). C) PSPC1 (paraspeckle component 1). *: p < 0.05.

    Single, B., et al. (1998). "Simultaneous release of adenylate kinase and cytochrome c in cell death." Cell

    Death Differ

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