Supplementary Information

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1 Supplementary Information Peroxiredoxin-2 and STAT3 form a redox relay for H 2 O 2 signaling Mirko C. Sobotta 1, Willy Liou 1, Sarah Stöcker 1, Deepti Talwar 1, Michael Oehler 1, Thomas Ruppert 2, Annette N. D. Scharf 2, and Tobias P. Dick 1 * 1 Division of Redox Regulation, DKFZ-ZMBH Alliance, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D Heidelberg, Germany 2 Core facility for mass spectrometry and proteomics, DKFZ-ZMBH Alliance, Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Im Neuenheimer Feld 282, D Heidelberg, Germany *Corresponding Author Tel.: Fax: t.dick@dkfz.de 1

2 Supplementary Results Supplementary Figure 1. Related to main Figure 1b. Prx2-containing high molecular weight conjugates are reducible with DTT. Figure 1b (left panel) shown together with the corresponding gel run under reducing conditions (right panel). Cells expressing either Prx1-SBP or Prx2-SBP were pulsed with 100 µm H 2 O 2 for 20 s. Affinity enriched complexes were separated under reducing conditions (25 mm DTT) and visualized by anti-sbp immunoblotting. Please note that Prx2 dimers were incompletely reduced (arrows). The asterisks indicate anti-sbp antibody background bands unrelated to Prx2. 2

3 Supplementary Figure 2. Related to main Figure 1c. Principle of 2D sequential non-reducing/reducing ( diagonal ) SDS-PAGE. (a) Protein mixtures containing disulfide conjugates are first separated by non-reducing SDS-PAGE. Thus, non-disulfide-linked proteins run according to their individual size, while disulfide-linked complexes run according to the combined size of their components. The lane is then cut out from the non-reducing gel and treated with DTT to reduce disulfides inside the gel matrix. (b) The DTT-treated lane is then placed horizontally on a reducing gel for separation in the second dimension. (c) A main diagonal is formed by non-disulfide-linked proteins, because they migrate the same distance in both dimensions. In contrast, proteins that were liberated from disulfideconjugates by DTT now migrate faster and thus appear below the main diagonal. Proteins liberated from the same disulfide-linked complex appear one below the other. 3

4 Supplementary Figure 3. Related to main Figure 1c. Identification of STAT3 by mass spectrometry. (a) Prx2 forms mixed disulfide conjugates with other proteins. Prx2-SBP was affinity enriched after pulsing cells with 100 µm H 2 O 2 for 20 seconds. Prx2-associated proteins were separated by non-reducing/reducing diagonal gel electrophoresis (explained in Supplementary Fig. 2) and visualized by silver staining. (b) A corresponding Coomassiestained gel was used for mass spectrometry. The gel slice yielding STAT3 peptides is indicated. (c) Positive identification of two STAT3 peptides by their LC-MS/MS fragmentation spectra. 4

5 Supplementary Figure 4. Expanded data set, related to main Figures 1d and 1e. (a) Specificity control for the co-precipitation of endogenous STAT3 with Prx2-SBP. The lack of non-specific binding of STAT3 or STAT3 conjugates to SA beads in lysates from cells lacking Prx2-SBP demonstrates Prx2-dependent co-precipitation of STAT3 (upper right panel). Prx2- SBP-expressing or empty vector-containing HEK293T cells were pulsed with 100 µm H 2 O 2 for 2 minutes before thiol blocking with 80 mm MMTS. (b) Figure 1d (right panel) combined with the corresponding input gel (left panel). (c) Figure 1e (upper part of upper right panel) with the corresponding input gel (left panels). In addition, the same membrane was stripped and reblotted for STAT3 to visualize STAT3 oxidation products (lower panels). All immunoblots are representative of n>3 independent experiments. 5

6 Supplementary Figure 5. Related to main Figure 2c. Ectopic expression of wild type Prx2 accelerates STAT3 oxidation in Prx2-depleted cells. Same experiment as shown in Fig. 2c, but cells were additionally transfected with either active Prx2-SBP or inactive Prx2(C51/172S)-SBP. Immunoblots are representative of n>3 independent experiments. 6

7 Supplementary Figure 6. Formation of Prx2-STAT3 disulfide-linked conjugates depends on the catalytic cysteines of Prx2 (a) HEK293T cells were transfected with wild type or mutant Prx2-SBP and untagged STAT3, and were pulsed with 100 µm H 2 O 2 for 2 minutes. Prx2-SBP affinity eluates were probed for STAT3 (left panel) and Prx2 (right panel). Please take note that ectopic Prx2-SBP is expressed in the presence of endogenous wild type Prx2. This allows formation of heterodimers between ectopic (wild type or mutant) Prx2 and endogenous wild type Prx2. Thus, the C P of endogenous Prx2 can form a disulfide with the C R of Prx2(C51S), and likewise, the C R of endogenous Prx2 can form a disulfide with the C P of Prx2(C172S). This explains why 7

8 Prx2(C51S), lacking C P, is capable of engaging in STAT3 thiol-disulfide exchange reactions. However, as expected, deletion of both cysteines (C R and C P ) completely prevents any disulfide exchange with STAT3. Interestingly, Prx2(C172S) forms increased levels of a STAT3- Prx2 1:1 disulfide-linked conjugate, suggesting the possibility that the C P sulfenic acid can directly condense with a target cysteine (Fig. 1a, upper branch). However, this finding does not exclude the possibility that wild type Prx2 first forms the C P -C R disulfide and then engages in thiol-disulfide exchange with STAT3 (Fig. 1a, lower branch). (b) The same samples after reduction with 50 mm DTT, resolved on 12% SDS-PAGE gels and probed for STAT3 (upper panel) and Prx2 (lower panel). All immunoblots are representative of n>3 independent experiments. 8

9 Supplementary Figure 7. Related to main Figure 4. Complete gels from which parts were used in the assembly of subfigures. (a) Complete gel used for the preparation of Figure 4c. (b) Complete gel used for the preparation of Figure 4e. (c) Complete gel used for the preparation of Figure 4f. All immunoblots are representative of n>3 independent experiments. 9

10 Supplementary Figure 8. Related to main Figure 4. A C-terminally truncated STAT3 variant lacking most of its TAD is resistant to Prx2-mediated oxidation. We created a truncated STAT3 variant (SBP-STAT3ΔTAD) similar to the naturally occurring STAT3 isoform by inserting a stop codon at position 716. SBP-STAT3 or SBP-STAT3ΔTADexpressing cells were pulsed with 50 µm H 2 O 2 for the indicated times before blocking thiols with 80 mm MMTS. Despite rapid Prx2 oxidation (upper panel), STAT3ΔTAD did not form covalent oligomers (lower panel). Immunoblots are representative of n>3 independent experiments. 10

11 Supplementary Figure 9. Related to main Figures 3 and 4. Prx2 mediated STAT3 oxidation leads to formation of an intramolecular disulfide bond between C712 and C718. SBP-tagged STAT3 was expressed in HEK293T cells which were or were not treated with 100 µm H 2 O 2 for 2 min, before thiol blocking with 100 mm NEM. Following SA affinity purification, STAT3 was subjected MS analysis. The 2-dehydro modification of the tryptic peptide containing C712 and C718 was highly enriched in monomeric STAT3 purified from H 2 O 2 treated cells relative to STAT3 purified from untreated cells. 11

12 Supplementary Figure 10. Related to main Figures 3 and 4. Analysis of STAT3 oxidation by differential alkylation. (a) The scheme summarizes the workflow for differential thiol labeling and data analysis. Please see the methods section for further details. (b) C712 and C718 can be oxidized independently of each other. (c) Oxidation of C426 is decreased in the absence of C418 and C

13 Supplementary Figure 11. Related to main Figure 5. Exogenously applied H 2 O 2 inhibits STAT3-dependent transcription. (a) H 2 O 2 treatment inhibits basal STAT3-dependent transcription from the SIE promoter. HEK293 cells transfected with a serum inducible element (SIE) firefly luciferase reporter construct and a CMV-driven Renilla luciferase construct were pulsed with increasing concentrations of H 2 O 2. Subsequently, cells were incubated for 8 h. In this assay we do not expect to see strong inhibition at low H 2 O 2 concentrations because low micromolar 13

14 concentrations are metabolized within minutes and thus will keep Prx2 oxidized only for a relatively short time period. In contrast, the assay read-out represents transcriptional activity over 8 h. Error bars indicate the standard deviation. RLU = relative light units. (b) H 2 O 2 treatment inhibits OSM-induced transcription from the SIE promoter. HEK293T cells were pre-treated with H 2 O 2 for 5 minutes. Subsequently the same cells were stimulated with 50ng/mL OSM and incubated for 8 h. The luciferase assay was performed as in a. (c) The experiment shown in b was repeated in Prx2-depleted cells. To compare the slope of the resulting curves, the signal at t=0 was set to 1 for both conditions. As expected, the inhibitory effect of H 2 O 2 was attenuated upon depletion of Prx2. We do not expect complete abrogation of H 2 O 2 -mediated transcriptional inhibition because the knockdown doesn t completely eliminate Prx2 activity, and the addition of H 2 O 2 to cells is likely to have secondary (indirect) effects, in particular phosphatase inhibition. Error bars indicate the standard deviation. (d-e) Similar to Prx2 depletion (Figs. 5e,f), expression of the catalytically inactive Prx2 mutant enhanced transcriptional responsiveness to OSM in HEK293T cells (d). A very similar effect was seen in the IL6-responsive cell line Huh7 (e). All assays were repeated at least three times. 14

15 Supplementary Figure 12. Related to main Figure 5. The TrxR inhibitor auranofin enhances Prx2 and STAT3 oxidation and inhibits STAT3 transcriptional activity. (a) TrxR inhibition leads to STAT3 oxidation in a time-dependent manner. SBP-STAT3- expressing HEK293T cells were pulsed with 100 µm H 2 O 2 for 2 min or treated with 1.5 µm auranofin (AFN) for the indicated times, before blocking thiols with 80 mm MMTS. (b) TrxR inhibition leads to STAT3 oxidation in a dose-dependent manner. Same experiment as in a, but cells were treated with increasing doses of AFN for 1 h. (c) TrxR inhibition attenuates cytokine-induced STAT3 phosphorylation. HEK293T cells were pre-treated for 1 h with AFN prior to cytokine stimulation (50 ng/ml) for 10 minutes. Free thiols were blocked with 80 mm MMTS. (d) TrxR inhibition attenuates cytokine-induced transcription from the serum inducible element (SIE) promoter in a concentration-dependent manner. HEK293T cells were transfected with a SIE Firefly luciferase reporter and a CMV-driven Renilla luciferase reporter. Cells were pre-treated with AFN for 30 minutes followed by stimulation with OSM (50 ng/ml) or IL6 and its receptor (50 ng/ml each) for 8 hours. Luciferase assays were performed in triplicates. Error bars indicate the standard deviation. RLU = relative light units. 15

16 Supplementary Figure 13. Related to main Figure 5. STAT3 forms a transient mixed disulfide intermediate with Trx1 subsequent to STAT3 oxidation. (a) Trx1-SBP and STAT3-Flag were co-expressed in HEK293T cells. Cells were treated with the indicated concentrations of H 2 O 2 for 3 minutes and 40 seconds. H 2 O 2 induced the formation of a 1:1 disulfide-linked conjugate between STAT3 and Trx1. (b) Re-probing of the same membrane with an anti-trx1 antibody confirms the upper band as a STAT3-Trx1 conjugate. (c) The appearance of the STAT3-Trx1 intermediate correlates with ongoing STAT3 reduction. Trx1-SBP expressing HEK293T cells were pulsed with 50 µm H 2 O 2 for the indicated times. Upper panel: Time course for the STAT3-Trx1 conjugate. Lower panel: Time course for STAT3 oligomers. The STAT3-Trx1 intermediate disappears when the STAT3 dimer is almost fully reduced. 16