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  • Molecular Cell, Volume 40

    Supplemental Information

    A Cytoplasmic ATM-TRAF6-cIAP1 Module Links Nuclear DNA Damage Signaling to Ubiquitin-Mediated NF-B Activation Michael Hinz, Michael Stilmann, Seda l Arslan, Kum Kum Khanna, Gunnar Dittmar, and Claus Scheidereit Inventory of Supplemental Information Hinz et al.

    Figure S1 and S2 are related to Figure 1 Figure S1 shows, that ATM translocation is also triggered by etoposide and is independent of the components of the PARP-1 signalosome. Figure S2 demonstrates selective calcium dependency of DNA damage induced NF-B activation and absence of early DNA damage induced alterations of calcium concentrations. Figure S3 is related to Figures 2 and 5 Figure S3 summarizes the results of an siRNA screen for pathway components required for DNA damage induced NF-B activation. Figure S4 is related to Figure 4 Figure S4 describes the requirement of TAK1 for DNA damage induced NF-B activation. Moreover it shows that TAK1 activation does not depend on PARP-1 or PIASy. Figure S5 is related to Figure 5 Figure S5 shows a longer exposure of figure 5B and calcium dependency of IKKgamma monoubiquitination Figure S6 is related to Figure 5 Figure S6 shows that the association of TRAF6 with cIAP1 depends on the RING domain in TRAF6. Figure S7 is related to Figure 6 Figure S7 shows that IKK monoubiquitination is induced by TNF.

  • Figure S1. DNA damage induced ATM translocation is independent of the PARP-1 signalosome, related to Figure 1. (A) HepG2 cells were treated with etoposide (10 M) for 30 min and fractionated. Crude cytoplasmic (CE) and nuclear extracts (NE) were analyzed by western blotting with ATM, P-ATM, PARP-1 or tubulin antibodies, respectively. (B) HepG2 cells were transfected with control siRNA or siRNA against PARP-1 or PIASy. Cells were treated with IR for 45 min, as indicated, and fractionated. Membranes and nuclear extracts (NE) were analyzed by western blotting with ATM, P-ATM, PARP-1 or PIASy antibodies, respectively. The membrane fraction was additionally analyzed with an IKK antibody.

    ATM

    P-ATM

    PARP-1

    PIASy

    IKK

    - -- -- - -- + ++ ++ + ++ IREto

    siCon

    trol

    siCon

    trol

    siPAR

    P-1

    siPAR

    P-1

    siPIAS

    y

    siPIAS

    y

    membranes NE

    270-

    270-

    170-

    170-

    130-130-

    72-

    55-

    55-

    ATM

    P-ATM

    PARP-1

    Tubulin

    CE NE

    BA

  • Figure S2. DNA damage- but not TNF-induced NF-B activation is calcium dependent, related to Figure 1. (A) HeLa cells were pre-incubated with solvent alone (DMSO) or with BAPTA for 60 min and treated with IR for the indicated time. WCE were monitored for NF-B activation by EMSA. Free DNA probe is not shown (B) Cells were treated as in (A). Crude cytoplasmic (CE) or nuclear (NE) fractions were analyzed by western blotting with antibodies against IB, p65, PARP-1 or p105. (C) Cells were pre-incubated with BAPTA for 60 min and

    A B

    C D

    DMSO DMSO

    DMSO

    DMSO

    DMSO

    DMSO

    BAPTA BAPTA

    BAPT

    A

    BAPT

    A

    BAPTA

    BAPTA

    0 0 00 0 01 1 11 1 12 2 22 2 2h post-IR h post-IR

    NF- B

    NF- B

    - - -- - -+ + ++ + +TNF (15 min) TNF (15 min)

    CE

    CE

    NE

    NE

    I B

    I B

    p65

    p65

    PARP-1

    p105

    p105

    43-

    43-

    34-

    34-

    130-

    130-

    130-

    72-

    72-

    IKK55-

    *

    *

    9

    11

    10

    12

    13

    min upon treatment 0 20 40 60

    DMSOATP

    9

    11

    10

    12

    Cal

    cium

    con

    cent

    ratio

    nar

    bita

    ry u

    nits

    (x10

    00)

    Cal

    cium

    con

    cent

    ratio

    nar

    bita

    ry u

    nits

    (x10

    00)

    min upon treatment 0 20 40 60

    DMSOEtoE

  • treated with TNF for 15 min. WCE were monitored for NF-B activation by EMSA as in (A). (D) Cells were treated as in (C). Crude cytoplasmic (CE) or nuclear (NE) fractions were analyzed by western blotting with antibodies against IB, p65, p105 and IKK. (E) Changes in intracellular Ca2+ concentration were assessed in WT MEFs upon treatment with 100 M etoposide (left panel) or with 1 mM ATP as a positive control (right panel). As a negative control the solvent DMSO was applied in each experiment. The fluorescence measurements were performed employing Fluo-4 NW Calcium Assay Kit (Invitrogen) and Mithras LB 940 plate reader (Berthold Technologies).

  • Figure S3. RNAi based NF-B pathway analysis, related to Figures 2 and 5. (A) Cells were transfected with siRNAs against NF-B signaling proteins as indicated. WCE were monitored for NF-B activation by EMSA. + transfection of two different siRNAs significantly reduced DNA damage induced NF-B activation, - no reduction, n.d. not determined. (B) HepG2 cells were transfected with siRNAs against RIP-1. Cells were treated with IR for 2 h. WCE were monitored for NF-B activation (EMSA) and immunoblotted for RIP-1 and PARP-1, respectively. (C) HepG2 cells were transfected with control siRNA or siRNA against cIAP1 and treated with IR for 2 h. WCE were monitored for NF-B activation (EMSA) and cIAP1 expression (WB).

    RNAi based NF- B pathway analysis*

    HeLa 293HepG2

    * readout: EMSA

    cIAP1 + n.d.+

    Ubc13 ++ +

    TAK1 ++ +

    TAB2 ++ +

    TRAF6 + + +

    MALT1 n.d. - -

    BCL10 n.d. - -

    TRAF2 - - -

    RIP1 - - -

    TAB1 - - -

    - + ++UT UT

    IRsiR

    IP1_

    1

    siRIP

    1-2

    NF- B

    72-

    EMSA

    WB

    130-

    RIP1

    PARP-1

    A

    B

    EMSA

    WB

    72-

    55-

    55-XIAP

    cIAP-1

    NF- B

    IR

    sicIAP

    1-1

    siCon

    trol

    - + +

    C

  • Figure S4. TAK1 requirement for DNA damage induced NF-B activation, related to Figure 4. (A) HepG2 cells were transfected with control siRNAs or siRNAs against TAK1. Cells were treated with IR (10 Gy) for 1 h. Cells were divided and WCE were monitored for NF-B activation (EMSA), while cytoplasmic extracts were immunoblotted for P-IKK, IKK, P-TAK1 and TAK1. (B) HeLa cells were transfected with control siRNAs or siRNAs against TAK1. Cells were treated with IR (10 Gy) for 1 h. Lysates were immunoprecipitated with IKK and analyzed for IKK kinase activity. Lysates were immunoblotted for TAK1 and IKK. (C) and (D) HepG2 cells were transfected with control siRNA or siRNAs against PARP-1 (C) or PIASy (D) and processed as in (A). Immunoblotting was performed with anti-P-TAK1, anti-TAK1, anti PARP-1 (C) or anti-PIASy (D). (E) HepG2 cells were treated with IR and fractionated. Membranes and cytosol were analyzed by western blotting as indicated.

    A

    B

    C

    D

    P-TAK1

    P-TAK1TAK1

    TAK1

    TAK1

    72-

    72-

    72-

    72-

    72-

    72-

    72-

    72-

    UT UT

    UT

    siTAK

    1_1

    siTAK

    1_1

    siPAR

    P-1-1

    siTAK

    1_2

    siPAR

    P-1-2

    siPIAS

    y

    siCon

    trol

    siRIP

    1

    siCon

    trol

    siCon

    trol

    siCon

    trol

    - -

    -

    --

    + + +

    +

    +

    + +

    -

    -

    + +

    +

    ++

    P-IKK

    PARP-1

    PIASy

    IKK

    P-TAK1

    TAK1

    EMSA

    EMSA

    EMSA

    WB

    WB

    WB

    WB

    IR IR

    IR

    IR

    95-

    95-

    130-

    *

    *

    *

    GST-I Bas1-53)

    (

    IKK

    KA

    55-

    membranes cytosol

    0 0 min post-IR

    72- TAK1

    10 1045 45

    E

    55-

    43-Tubulin

    55-

    72-TNFR

    72- P-TAK1

    TAB272-

    55- TAB1

    NF- B

    n.s.

    NF- B

    n.s.

    NF- B

    n.s.

  • Figure S5. DNA damage induced IKK monoubiquitination takes place in the cytoplasm, related to Figure 5. (A) Long exposure of the experiment in figure 5B. (B) HepG2 cells were pre-incubated with solvent alone (DMSO) or with BAPTA for 60 min, treated with IR for 45 min, lysed and immunoprecipitated with anti-IKK. Lysates and IP extracts were immunoblotted with IKK antibodies.

    - -+ + IR

    *55-

    DMSO BAPTA

    55-

    minpost-IR00 2020 4040 6060

    IP: IKKWB: IKK

    IP: IKKWB: IKK

    A BCE NE

    *

  • Figure S6. DNA damage induces an interaction between TRAF6 and cIAP1, related to Figure 5. 293 cells, transfected with FLAG-TRAF6-WT or FLAG-TRAF6, a deletion mutant lacking the RING domain, were either left untreated or exposed to IR for 40 min. Cytoplasmic extracts were immunoprecipitated with anti-FLAG. Lysate and IP extracts were immunoblotted with cIAP1 and TRAF6 antibodies.

    0 00 040 40 40 40 time upon IR40 40

    IP FLAG

    IP FLAG

    TRAF6

    72 -

    26 -

    43 -

    72 -

    cont

    rol

    cont

    rol

    55 -

    34 -

    55 -

    cIAP1

    T6-WT T6-WTT6- T6-

    Input

    *

  • Figure S7. TNF signaling induces IKK ubiquitination, related to Figure 6. HepG2 cells were treated with TNF for the indicated time. Lysates were immunoprecipitated with IKK antibody and immunoblotted with antibodies against IKK and Ubiquitin (Ub).

    0 3 5 10 15 0 3 5 10 15

    Input IP: IKKmin

    TNF

    55-

    55-

    IKK

    Ub

    *

    *

  • Supplemental Experimental Procedures

    Cell culture

    MEFs were maintained in Hepes-buffered Dulbecco's modified Eagle's medium-Glutamax-I

    (Invitrogen) containing 4.5 g/l glucose, 10% fetal calf serum