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1 doi: /nture09663 Scrmle shnlrp3 shcsp1 IL-1β (p17) IL-1β (pg/ml) Wt Nlrp3-/- Ipf-/- 0 APDC IL-1β (p17) Supplementl Figure 1. Mitochondril ROS cn trigger NLRP3 inflmmsome ctivtion, THP1 cells stly expressing shrna ginst the indicted trget genes or LPS-primed one mrrow-derived mcrophges (BMDMs) from wild type (wt), Nlrp3 or Ipf deficient mice were stimulted 6 hrs with MSU (150 μg/ml), rotenone (10 μm for THP1 cells nd 40 μm for BMDMs) or ntimycin (40 μg/ml for THP1 cells nd 10 μg/ml for BMDMs). The relese Il- 1 ws then determined western lot (THP1) or ELISA (BMDMs)., THP1 cells were stimulted for 6 hrs with MSU (150 μg), rotenone (10 μm) or ntimycin (40 μg/ml) in the presence of APDC (100 μm) or its sence () nd the relese of IL-1 determined WWW NATURE.COM/NATURE 1

2 IL-β (p17) Norlmlized LDH relese IL-β (p17) Norlmlized FL2/FL1 rtio of JC-1 fluorescence No stining CCCP 50 um CCCP 5 um Mendione Rotenone Mitosox Supplementl Figure 2. Inhiition of mitochondril complex I nd complex 3 ctivity results in NLRP3 inflmmsome ut not in cell deth., THP-1 cells were stimulted with MSU (150 μg/ml), rotenone (10 μm), ntimycin (40 μg/ml) for 6 h. IL-1 nd LDH relese were determined., THP-1 cells were stimulted with the respirtory chin uncoupler CCCP or with the pro-oxidnt mendione, rotenone or MSU s controls. The mitochondril memrne potentil nd ROS genertion were mesured y FACS using the fluorescent proes JC-1 nd mitosox, respectively WWW NATURE.COM/NATURE 2

3 unstined 3-MA unstined Scrmle shbeclin-1 shatg5 MitoSOX MSU IL-1β (p17) c MSU 3-MA (mm) 5 10 MSU 3-MA (mm) 5 10 IL-1β (p17) Pro-IL-1β APDC d Norlmlized LDH relese MSU Nigericin Lysis Supplementl Figure 3. Inhiition of utophgy/mitophgy genertes ROS, ctivtes the NLRP3 inflmmsome which is inhiited y the nti-oxidnt APDC ut does not induce cell deth., THP1 cells stimulted with 3-Methyldenine (3-MA, 10 mm) for 24 h or THP1 cells stly expressing shrna ginst Beclin-1 or ATG5 were stined with MitoSOX for 30 min nd nlyzed y flow cytometry., THP1 cells were stimulted for 6 hrs with MSU (150 μg/ml) or the indicted mounts of 3-MA, superntnts nd cell extrcts were nlyzed y western lotting. c, THP-1 cells were treted with 3-MA in the presence or sence of APDC. d, THP-1 cells stly expressing shrna ginst Beclin-1 or ATG5 were stimulted with MSU, nigericin for 6 hrs nd cell deth determined y the mesurement of LDH relese. WWW NATURE.COM/NATURE 3

4 DAPI Flg-NLRP3 Clreticulin Merge DAPI Flg-ASC Clreticulin Merge DAPI Flg-NLRP3 Mitotrcker Merge DAPI Flg-ASC Mitotrcker Merge DAPI Flg-NLRP3 Gintin Merge DAPI Flg-ASC Gintin Merge DAPI Flg-NLRP3 Lysotrcker Merge DAPI Flg-ASC Lysotrcker Merge Supplementl Figure 4. Locliztion of NLRP3 () nd ASC () in unstimulted THP-1 cells stly expressing Flg-NLRP3 or Flg-ASC. Orgnelle mrkers used re clreticulin (ER), mitotrcker (mitochondri), Gintin (golgi) nd lysotrcker (lysosomes). WWW NATURE.COM/NATURE 4

5 Isotype control 3d20 Supplementl Figure 5. Locliztion of NLRP3 in unstimulted THP-1 cells stly expressing Flg-NLRP3. Arrows point to immuno-gold leled NLRP3. Isotype control is shown in the lower right pnel WWW NATURE.COM/NATURE 5

6 Flg-NALP3 Clreticulin Mitotrcker Merge Nigericin MSU Nigericin MSU Flg-ASC Clreticulin Mitotrcker Merge Supplementl Figure 6. Upon stimultion of the NLRP3 inflmmsome, NLRP3 nd ASC co-loclize with perinucler orgnelles tht stin positively for oth, the ER-mrker clreticulin nd mitochondri-ssocited mitotrcker. THP-1 cells expressing Flg-NLRP3 () or Flg-ASC () were nlyzed. WWW NATURE.COM/NATURE 6

7 MSU Scrmle Scrmle shvdac1- shvdac1- shvdac2- shvdac2- shvdac3- shvdac3- IL-1 (p17) Pro-IL-1β VDAC1 VDAC2 VDAC3 GAPDH IL-1β VDAC1 Scrmle shvdac1 c IL-1β Scrmle shvdac3 Supplementl Figure 7. VDACs re importnt for NLRP3- ut not AIM2- or IPAFinflmmsome ctivtion., Inhiition of NLRP3 inflmmsome ctivtion y MSU in vrious THP-1 clones expressing shrna trgeting one of the 3 VDAC isoforms., ctivtion of the AIM2 nd the IPAF proceeds normlly in the sence of VDAC1. c, The sence VDAC3 hs no effect on NLRP3 inflmmsome ctivity. WWW NATURE.COM/NATURE 7

8 MSU R837 Nigericin Scrle DAPI Mitosox shvdac2 shvdac1 Supplementl Figure 8. Inhiition of mitochondril ROS in THP-1 cells stly expressing VDAC1 nd VDAC2 upon stimultion wth MSU, R837 or nigericin. WWW NATURE.COM/NATURE 8

9 IL-1 β (pg/ml) LPS MSU (μg) Alum (μg) Nigericin (μm) Slmonell (dilution) TNF (pg/ml) wt Bcl-2-tg LPS WWW NATURE.COM/NATURE 9

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