Microtubule-driven spatial arrangement of mitochondria promotes activation of the NLRP3 inflammasome

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1 Supplementry Informtion Microtuule-driven sptil rrngement of mitochondri promotes ctivtion of the NLRP3 inflmmsome Tkum Misw 1,2, Michihiro Tkhm 1,2, Ttsuy Kozki 1,2, Hnn Lee 1,2, Jin Zou 1,2, Ttsuy Sitoh 1,2,, Shizuo Akir 1,2, Nture Immunology doi:1.138/ni.255

Supplementry Figure 1 IL-1β (ng/ml) Cell Viility (ritrry units) IL-1β (ng/ml).1.5.1.5.1.5 1.

DMSO C2 : Podophyllotoxin C3 : Nocodzole C4 : Colchicine Silic Silic, LAMP1, Nuclei Supplementry Figure 1.

(,) Primed J774 cells were pretreted with the chemicl compound lirry nd stimulted with nigericin, flgellin or Poly(dAdT). IL-1β production () nd cell viility () were mesured.

The culture superntnts were nlysed y ELISA. (d) Primed BMMs were treted with incresing dose of colchicine (5-15 µm) or nocodzole (5-15 µm).

2 Supplementry Figure 1 IL-1β (ng/ml) Cell Viility (ritrry units) IL-1β (ng/ml) Nocodzole C1 C2 C3 C4 C1 C2 C3 C4 ATP Silic (NLRP3) (NLRC4) (AIM2) (NLRP3) (NLRC4) (AIM2) c d e Colchicine NLRP3 ASC Actin Pm3CSK4 Colchicine Nocodzole C1 : DMSO C2 : Podophyllotoxin C3 : Nocodzole C4 : Colchicine Silic Silic, LAMP1, Nuclei Supplementry Figure 1. Screening of chemicl compounds cple of regulting inflmmsome ctivtion. (,) Primed J774 cells were pretreted with the chemicl compound lirry nd stimulted with nigericin, flgellin or Poly(dAdT). IL-1β production () nd cell viility () were mesured. (c) Primed one-mrrow-derived mcrophges (BMMs) were pretreted with incresing doses of nocodzole (5-15 µm) nd then stimulted with the indicted lignds. The culture superntnts were nlysed y ELISA. (d) Primed BMMs were treted with incresing dose of colchicine (5-15 µm) or nocodzole (5-15 µm). The cells were then nlyzed y immunolotting with ntiodies for the indicted proteins. (e) Primed BMMs were pretreted with colchicine (1 µm) nd incuted with rhodmine-silic. The cells were then fixed nd nlysed y immunocytochemistry. Red, green nd lue signls indicte rhodmine-silic, LAMP1 nd nuclei, respectively. The experiments were repeted t lest three times. The results re represented s the men ± s.d. of three independent smples. The scle r represents 1 µm. Nture Immunology doi:1.138/ni.255

(mitochondril protein) nd clreticulin (ER

3 Supplementry Figure 2 Tom2 Clreticulin Merge PL-positive dots Actin filments Nuclei Supplementry Figure 2. Sucellulr locliztions of mitochondri nd the ER in BMMs stimulted with, flgellin or. () Primed BMMs were stimulted with the indicted lignds. The cells were then fixed nd nlysed y immunocytochemistry. The sucellulr locliztions of Tom2 (mitochondril protein) nd clreticulin (ER protein) re shown. The sterisks indicte nuclei. () Primed BMMs were stimulted with the indicted lignds, nd then sujected to PL ssys using mouse nd rit isotype IgGs. Red, green nd lue signls indicte PL-positive signls, ctin filments nd nuclei, respectively. The experiments were repeted t lest three times. The scle r represents 1 µm. Nture Immunology doi:1.138/ni.255

4 Supplementry Figure 3 Ciliorevin-D Tom2 α-tuulin Merge Tom2 α-tuulin Merge Supplementry Figure 3. Suppression of nigericin- or -induced mitochondril trnsport y ciliorevin-d. Primed BMMs were pretreted with ciliorevin-d (1 µm), stimulted with the indicted lignds, nd then nlysed y immunocytochemistry. The sucellulr locliztions of Tom2 nd α-tuulin re shown. Red, green nd lue signls indicte Tom2, α-tuulin nd nuclei, respectively. The experiments were repeted t lest three times. The scle r represents 1 µm. Nture Immunology doi:1.138/ni.255

5 Supplementry Figure 4 IL-1β (ng/ml) Tom2 Clreticulin Merge IL-1β (ng/ml) ATP Silic ATP Silic c PL-positive signls, Actin filments, Nuclei d PL-positive cells (%) Supplementry Figure 4. K + -efflux-independent nd ROS-independent ggregtion of mitochondri t perinucler region. () Primed BMMs were pretreted with incresing dose of extrcellulr high K + (5-25 mm) or (1-5 µm), nd then stimulted with the indicted lignds. The culture superntnts were nlysed y ELISA. () Primed BMMs were pretreted with extrcellulr high K + (25 mm) or (5 µm), stimulted with the indicted lignds, nd then nlysed y immunocytochemistry. The sucellulr locliztions of Tom2 nd clreticulin re shown. Red, green nd lue signls indicte Tom2, clreticulin nd nuclei, respectively. (c,d) Primed BMMs were pretreted with extrcellulr high K + (25 mm) or (5 µm) nd then stimulted with nigericin or. The cells were nlysed y PL ssys ginst ASC nd NLRP3 (c). The percentges of BMMs with PL-positive signls were clculted (d). The experiments were repeted t lest three times. The results re represented s the men ± s.d. of three independent smples. The scle r represents 1 µm. ; not detected. Nture Immunology doi:1.138/ni.255

Supplementry Figure 5 Colchicine % 13.8% 12.% SSC-H 29.9% 14.6% 28.5% MitoSOX Supplementry Figure 5. Unltered ROS genertion y inhiition of tuulin polymeriztion.

6 Supplementry Figure 5 Colchicine % 13.8% 12.% SSC-H 29.9% 14.6% 28.5% MitoSOX Supplementry Figure 5. Unltered ROS genertion y inhiition of tuulin polymeriztion. Primed BMMs were pretreted with (5 µm) or colchicine (1 µm), nd stimulted with nigericin. Cells were stined with MitoSox nd then nlysed y flow cytometry. The experiments were repeted t lest three times. Nture Immunology doi:1.138/ni.255

7 Supplementry Figure 6 MEC-17 mrna (Fold reduction) 1..5 Control MEC-17 Ac-α-Tuulin Poly E-α-Tuulin Tyr-α-Tuulin α-tuulin Control MEC-17 Supplementry Figure 6. Involvement of MEC-17 in the genertion of cetylted α-tuulin in J774 cells. () The levels of MEC-17 mrna expression in J774 cells stly expressing MEC-17 or control were quntified y quntittive RT-PCR. () The post-trnsltionl modifictions of α-tuulin in J774 cells stly expressing the MEC-17 or control were nlysed y immunolotting. The experiments were repeted t lest three times. The results re represented s the men ± s.d. of three independent smples. Nture Immunology doi:1.138/ni.255

Supplementry Figure 7 c SIRT2 mrna (Fold reduction) 1.

Inhiitor III AGK2 - + - + d e Ac-α-Tuulin α-tuulin Merge Tom2 Clreticulin Merge

Involvement of SIRT2 in the genertion of cetylted α-tuulin in J774 cells.

control were quntified y quntittive RT-PCR.

hours, nd then nlysed y immunolotting (c) nd immunocytochemistry (d,e) with

8 Supplementry Figure 7 c SIRT2 mrna (Fold reduction) 1..5 Control SIRT2 Ac-α-Tuulin α-tuulin Control SIRT2 Ac-α-Tuulin α-tuulin SIRT1 Inhiitor III AGK d e Ac-α-Tuulin α-tuulin Merge Tom2 Clreticulin Merge SIRT1 Inhiitor III SIRT1 Inhiitor III No Pre-Tx No Pre-Tx Supplementry Figure 7. Involvement of SIRT2 in the genertion of cetylted α-tuulin in J774 cells. () The levels of SIRT2 mrna expression in J774 cells stly expressing the SIRT2 or control were quntified y quntittive RT-PCR. () The level of cetylted α-tuulin in J774 cells stly expressing the SIRT2 or control were nlysed y immunolotting. (c-e) Primed BMMs were treted with SIRT1 inhiitor III (1 µm) or AGK2 (1 µm) for 2 hours, nd then nlysed y immunolotting (c) nd immunocytochemistry (d,e) with ntiodies for the indicted proteins. The experiments were repeted t lest three times. The results re represented s the men ± s.d. of three independent smples. The scle rs represent 1 µm. Nture Immunology doi:1.138/ni.255

9 Supplementry Figure 8 NAD + reltive control (ritrry units) 1..5 IL-1β (ng/ml) ABA (-) (-) 3-ABA (-) (-) Supplementry Figure 8 PARP-1-independent reduction of NAD + induced y NLRP3-inflmmsome inducers. (,) Primed BMMs were pretreted with incresing dose of 3-ABA (1-1 µm) nd then stimulted with nigericin. The intrcellulr NAD + level () ndil-β production () were mesured. Ech NAD + level in BMMs ws divided y the NAD + level in untreted BMMs to determine the NAD + reltive control (ritrry units). The experiments were repeted t lest three times. The results re represented s the men ± s.d. of three independent smples. ; not detected. Nture Immunology doi:1.138/ni.255

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION doi:10.1038/nture09663 Scrmle shnlrp3 shcsp1 IL-1β (p17) IL-1β (pg/ml) 2000 1500 1000 500 Wt Nlrp3-/- Ipf-/- 0 APDC IL-1β (p17) Supplementl Figure 1. Mitochondril ROS cn trigger NLRP3 inflmmsome ctivtion,