WT Xid (h) poly(da:dt) Alum. Poly(dA:dT) 0.05
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1 IL1 (ng/ml) (1 x 1 3 cells) DMSO LFMA13 R46 IL1 (ng/ml) Control shrna TNFa (ng/ml) Alum Nigericin ATP IL1 (ng/ml) IL1 (ng/ml) IL1 (ng/ml) a c f Sup DMSO Alum/DMSO Alum/LFMA13 *** ** () LPS Poly(I:C) Neutrophils WT Xid WT Xid PBS Alum IL1 p17 Casp1 p WT Xid (h) Macrophages *** *** 4 Alum WT Xid WT Xid PBS Alum MSU Poly(dA:dT) () LPS LPS LFMA13 (mm) d 6 4 LPS LFMA13 *** * Supplementary Figure 1. is involved in activation of inflammasome, ut not AIM2 inflammasome. (a) ELISA of murine IL1β, TNFa in supernatants of LPS or poly(i:c)primed murine peritoneal macrophages that were pretreated with LFMA13 and then stimulated with alum for 3 h. () ELISA of murine IL1β in supernatants of LPSprimed peritoneal macrophages from Xid and WT mice stimulated with alum up to h. (c) Flow cytometric analysis of Gr1 F4/8 neutrophils and F4/8 monocytesmacrophages in the peritoneal cavity from Xid (n = 8) or WT (n = 7) mice that were injected with alum for 15 h. (d) ELISA of murine IL1β in supernatants of LPSprimed BMDCs that were pretreated with LFMA13 and then stimulated with the indicated inflammasome activators for 3 h. (e) ELISA of human IL1β in supernatants of THP1Mfs that were pretreated with LFMA13 or R46 and then stimulated with poly(da:dt) for 3 h. (f) Immunolot analysis of human IL1β p17 and caspase1 p in supernatants of LPSprimed human lood monocytes that were pretreated with LFMA13 and then stimulated with the indicated inflammasome activators for 3 h. (g) ELISA of human IL1β in supernatants of knockdown and control THP1Mfs stimulated without or with alum or poly(da:dt) for 3 h. Data are representative of three independent experiments. Data are presented as mean ± SD (triplicate). *, P <.3; **, P <.1; ***, P <.3. Twosided Student s ttest. (h) Immunolot analysis of human and a tuulin in knockdown and control THP1 cells. Lower numers indicate the protein expression ration of to atuulin. e g poly(da:dt) No stimuli THP1 Mf ***.1.5 Alum ** h atuuin Control shrna * Poly(dA:dT)
2 a Crosslink Oligomer Solule Dimer Monomer Insolule Flag T7 HA LFMA13 py Flag T7 HA HADTK LFMA13 Supplementary Figure 2. overexpression promotes oligomerization and aggregation in HEK 293T cells. (a) Immunolot analysis of human,, and in the BS3treated or untreated cell lysates of HEK 293T cells that were transfected with Flag, T7, or HA. Six hours after the transfection, LFMA13 was added to the cell culture. () Immunolot analysis of human,, and in the Triton Xsolule and insolule fractions of HEK 293T cells transfected with Flag, T7, HAfulllength, or HADTK. Six hours after the transfection, LFM A13 was added to the cell culture. Data are representative of three independent experiments.
3 WT R28C R37G R525Q E567K WT R28C R37G R525Q E567K a R28C PH TH SH3 SH2 TK R37G R525Q E567K HA () (py223) Solule Insolule HA () HA () Nigericin Supplementary Figure 3. XLAtype mutations impede redistriution. (a) Schematic of human and four XLAtype mutations (Left) and immunolot of phosphorylation of Tyr223 of human in THP1Mfs staly expressing HA (WT or XLA mutant) (Right). () Immunolot analysis of in the Triton Xsolule and insolule fractions in THP1Mfs staly expressing HA (WT or XLA mutant) stimulated with nigericin for 3 min. Data are representative of two independent experiments.
4 Ca 2 ATP DMSO LFMA13 LeuLeuOMe Ca 2 (sec) Supplementary Figure 4. LFMA13 does not inhiit Ca 2 influx. Fluo4 AMloaded THP1 cells were treated with LFMA13, then stimulated with ATP or LeuLeuOMe. The fluorescent intensity was measured using flow cytometric analysis. Data are representative of two independent experiments.
5 LPS Alum LFMA (min) (min) PLCg1 pplcg1 PLCg2 pplcg2 JNK pjnk p38 pp38 Supplementary Figure 5. LFMA13 does not inhiit PLCg phosphorylation. Immunolot analysis of PLCg1, PLCg2, JNK, p38 and the phosphorylated form of each molecules in LPSprimed peritoneal macrophages that were pretreated with LFMA13 and then stimulated with alum for 3 or 18 min.
6 a Unstain DMSO LFMA13 Unstain WT Xid MitoSOX MitoSOX Supplementary Figure 6. is involved in mitochondrial ROS production. MitoSOXloaded THP1 cells treated with LFMA13 (a) or peritoneal macrophages from WT (CBA/J) or Xid (CBA/N) mice () were stimulated with ATP. The fluorescent intensity was measured using flow cytometric analysis. Data are representative of two independent experiments.
7 IL1 (ng/ml) IL1 (ng/ml) a 4 THP1Mf THP1Mf Sup IL1 p17 Casp1 p ProIL1 Procasp1 () Alum Alum Poly(dA:dT) LFMA13 Supplementary Figure 7. overexpression induces inflammasome activation in THP1macrophages. ELISA of human IL1β in supernatants (a,) and immunolot analysis of IL1β and caspase1 in supernatants and cell lysates (a) of THP1 Mfs and THP1Mfs staly expressing HA that were stimulated with alum or poly(da:dt) for 3 h. Data are representative of three independent experiments. Data are presented as mean ± SD (triplicate).
8 d GFP Flag FlagPYD FlagNACHT FlagLRR HA IP:aFlag IB:aHA IB:aFlag a TH PH SH3 SH2 TH PH SH3 SH2 TK TH SH3 SH2 TK HA HADPH HADTK GFP Flag Flag HA HADPH HADTK IB:aHA IB:aFlag IP:aFlag # TH PH SH3 SH2 TK PH SH3 SH2 TK HA HAPH HASH2/3 HATK GFP Flag Flag HA HAPH HASH2/3 HATK IP:aFlag IB:aHA IB:aFlag # Supplementary Figure 8. Determination of human domains that ind to and. Coimmunoprecipitation and immunolot assays of HA (Fulllength or DPH or DTK domains) and Flag or (a), HA (Fulllength or PH, SH2/3, or TK domains) and Flag or (), HA and Flag (Fulllength, PYD, or CARD) (c), or HA and Flag (Fulllength, PYD, or NACHT or LRR domains) (d) from HEK293T cells cotransfected with plasmids expressing the indicated molecules. #, Ig light chains (a,). Data are representative of two independent experiments. c IP:aFlag GFP Flag FlagPYD FlagCARD HA IB:aHA IB:aFlag
9 Infract volume (mm 3 ) Infract volume (mm 3 ) Infract volume (mm 3 ) a 5 4 ** WT Il1 / 5 4 Il1 / PBS Irutini Day3 Day3 c Il1 / PBS Irutini Day3 Supplementary Figure 9. IL1 is responsile for ischemic rain injury. (a) Infarct volume on day 3 after stroke onset in WT or Il1 / mice (n = 8 for WT; n = 7 for Il1 / ). (, c) Infarct volume on day 3 after stroke onset in Il1 / mice treated with PBS or irutini (3.125 mg/kg/day on Day, 1) immediately after 6 min () or 9 min (c) occlusion followed y reperfusion (n = 6 for PBS; n = 5 for irutini). Scale ars, 1 mm. Data are presented as mean ± SEM. **, P <.1. Twosided Student s ttest.
10 F4/8 CD45 (1 x 1 4 cells) 3 Microglia 2 CD11 CD45 high CD11 high 1 Macrophages Ischemia Irutini Neutrophils Macrophages Microglia Neutrophils Gr1 Supplementary Figure 1. The numers of neutrophils and macrophages are not affected y irutini. The numers of neutrophils, macrophages, and microglia in the rain were counted y FACS on day 1 after stroke onset (n = 4). Data are presented as mean ± SEM. Twosided Student s ttest. Left panels show representative FACS profiles of mononuclear cells from the ischemic rain.
11 IL1 (pg/ml) Infract volume (mm 3 ) Infract volume (mm 3 ) a ** * c Supplementary Figure 11. Mature IL1 is reduced in Xid. (a) ELISA of IL1 in rain from WT (CBA/J) or Xid (CBA/N), mice on day 1 after stroke onset (n = 3). () Infarct volume on day 4 after stroke onset in WT, Xid mice, or Xid mice transferred with CD19 B cells (n = 11 for WT; n = 8 for Xid; n = 4 for Xid B cell). (c) Infarct volume on day 3 after stroke onset in Xid mice treated with PBS or irutini (3.125 mg/kg/day on Day ) immediately after stroke onset (n = 5 for PBS; n = 7 for irutini). Data are presented as mean ± SEM. *, P <.3; **, P <.1. Twosided Student s ttest.
12 Alum MSU Nigericin ATP LeuLeuOMe Fig. 1a IL1 p17 Fig. 1d Casp1 p Fig. 1h IL1 p17 47 Casp1 p/p22 ProIL1 47 Casp1 p CBA/J CBA/N 47 ProIL1 ProIL1 IL1 p17 Procasp1 atuulin Procasp1 47 Fig. 1g Casp1 p 47 LPS LFMA13 LPS LFMA13 Supplementary Figure 12. Fulllength images of Western lots presented in the main figures.
13 Others Microglia Macrophages Fig. 2a Fig. 2d Fig. 2e Crosslink Oligomer Dimer Solule (py223) LPS Nigericin LFMA13 Monomer Flag WT Flag Y146F T7 HA Insolule IP: a IP: a Fig. 2c LPS ATP Poly(dA:dT) LFMA13 Solule Insolule Fig. 4d p (Y223) atuulin PMA Alum LFMA13 Supplementary Figure 12. Fulllength images of Western lots presented in the main figures.
14 Supplementary Tale 1. List of pharmacological inhiitors. Inhiitor Company Target Final conc. (mm) LFMA13 Cayman Irutini ShanHai Biochempartner 1 HIF1 inhiitor Caliochem HIF1 5 ASN11542 Caliochem PKR 5 Chk2 inhiotr II Caliochem CHK2 2 Calpeptin Caliochem Calpain 5 KN62 Caliochem CAMK2 4 SB358 Caliochem p38 1 R46 ShangHai Biochempartner Syk 1
15 Supplementary Tale 2. The percentages of cereral lood flaw reduction after common carotid artery (CCA) and middle cereral artery (MCA) occlusion (mean ± SD). n Before After CCA MCA WT (C57BL/6J) PBS ± ±2.3 Irutini ± ±3.2 Il1 / (C57BL/6J) PBS ± ±3.6 Irutini ± ±1.9 WT (CBA/J) PBS ± ±4.2 PBS ± ±4.5 Xid (CBA/N) Irutini ± ±4.8 B cell transfer ± ±4.2
Type of file: PDF Title of file for HTML: Supplementary Information Description: Supplementary Figures
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Supplementary information
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Supplementary Figure 1 AAV-GFP injection in the MEC of the mouse brain C57Bl/6 mice at 4 months of age were injected with AAV-GFP into the MEC and sacrificed at 7 days post injection (dpi). (a) Brains
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Supplementary Figure 1 a γ-h2ax MDC1 RNF8 FK2 BRCA1 U2OS Cells sgrna-1 ** 60 sgrna 40 20 0 % positive Cells (>5 foci per cell) b ** 80 sgrna sgrna γ-h2ax MDC1 γ-h2ax RNF8 FK2 MDC1 BRCA1 RNF8 FK2 BRCA1
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Supplementary Figure S A) The blots shown in Figure B were qualified by using Gel-Pro analyzer software (Rockville, MD, USA). The ratio of LC3II/LC3I to actin was then calculated. The data are represented
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a 1% 5% % 1% 5% % OVAb βgal activity A.U. (x1 4 ) 2 1 5 βgal activity A.U. (x1 4 ) 2 1 BSAb 2 hours 4 hours OVAb BSAb OVAb BSAb,1 1 1 1 SIINFEKL (ng/ml) CFSE b Beads Alexa488 (%) 8 6 4 2 ** ** 1:1 5:1
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