Supplemental Information. The Pore-Forming Protein Gasdermin D Regulates. Interleukin-1 Secretion from Living Macrophages

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Immunity, Volume 48 upplemental Information The PoreForming Protein Gasdermin D Regulates Interleukin1 ecretion from Living acrophages Charles L. Evavold, Jianbin Ruan, Yunhao Tan, hiyu Xia, Hao Wu, and Jonathan C. Kagan

A B C Gsdmd / clone 1 Gsdmd / clone 2 Nonspecific GDDc1 βactin 1 9 8 7 6 5 4 3 2 1 Gsdmd / 1 µ Nigericin 1 µ Nigericin IL1β (pg/ml) 14 12 1 8 6 4 2 Gsdmd / 1 µ Nigericin 1 µ Nigericin D E F 1 9 8 7 6 5 4 3 2 1 15 3 45 6 75 9 15 12 Time (min) Untreated 1 µ Nigericin 1 µ Nigericin Gsdmd / Untreated Gsdmd / Gsdmd / 1 µ Nigericin Gsdmd / 1 µ Nigericin PI (% aximum) TNFα (pg/ml) 2 15 1 5 Glycine only Gsdmd / Glycine 1 µ Nigericin 1 µ Nigericin Glycine 1 µ Nigericin 1 µ Nigericin Glycine 1 9 8 7 6 5 4 3 2 1 Gsdmd / 5 m K 5 m K 1 µ Nigericin 1 µ Nigericin 5 m K 1 µ Nigericin 1 µ Nigericin 5 m K G H I IL1β (pg/ml) 14 12 1 8 6 4 2 Gsdmd / 5 m K 5 m K 1 µ Nigericin 1 µ Nigericin 5 m K 1 µ Nigericin 1 µ Nigericin 5 m K PI (% aximum) 1 9 8 7 6 5 4 3 2 1 15 3 45 6 75 9 15 12 Time (min) Untreated 5 m K 5 m K 1 µ Nigericin 1 µ Nigericin 5 m K 1 µ Nigericin 1 µ Nigericin 5 m K Gsdmd / Untreated Gsdmd / 5 m K Gsdmd / Gsdmd / 5 m K Gsdmd / 1 µ Nigericin Gsdmd / 1 µ Nigericin 5 m K Gsdmd / 1 µ Nigericin Gsdmd / 1 µ Nigericin 5 m K TNFα (pg/ml) 2 15 1 5 Gsdmd / PA PA Glycine PA PA Glycine LFnFla LFnFla Glycine LFnFla LFnFla Glycine PA LFnFla PA LFnFla Glycine PA LFnFla PA LFnFla Glycine Figure 1

Figure 1: Characterization of GDD deficient macrophages. Related to Figure 1. (A) Immunoblot validation of GDD deficiency in two clonal populations after Cas9targeting using two independent grnas to Gsdmd. (B) LDH cytotoxicity measurements of cellfree culture supernatants. and Gsdmd/ clone 2 ibds were primed with for 3 hours (or not), and then treated with nigericin for 2 hours. (C) IL1β ELIA measurements of cellfree culture supernatants. and Gsdmd/ clone 2 ibds were primed with for 3 hours (or not), and then challenged with nigericin for 2 hours. (D) PI real time membrane permeability kinetic time course of live cells. and Gsdmd/ clone 2 ibds were primed with for 3 hours (or not), and then challenged with nigericin for 2 hours. PI was added to assay membrane permeability over time after priming and secondary stimulation. (E, I) TNFα ELIA measurements of cellfree culture supernatants. and Gsdmd/ clone 1 ibds were primed with for 3 hours (or not), and then challenged with nigericin for 2 hours or Flatox (2 µg/ml PA and.5 µg/ml LFnFla) for 2 hours. timulations contained a final concentration of m Glycine or 5 m Glycine. (F) LDH cytotoxicity measurements of cellfree culture supernatants. and Gsdmd/ clone 1 ibds were primed with for 3 hours (or not), and then challenged with nigericin for 2 hours. timulations contained an additional concentration of m K or 5 m K. (G) IL1β ELIA measurements of cellfree culture supernatants. and Gsdmd/ clone 1 ibds were primed with for 3 hours (or not), and then challenged with nigericin for 2 hours. timulations contained an additional concentration of m K or 5 m K. (H) PI real time membrane permeability kinetic time course of live cells. and Gsdmd/ clone 1 ibds were primed with for 3 hours (or not), and then challenged with nigericin for 2 hours. timulations contained an additional concentration of m K or 5 m K. PI was added to assay membrane permeability over time after priming and secondary stimulation.

Data with error bars are represented as mean ± E. Each panel is a representative experiment of at least 3 repeats.

A B C D 1 9 8 7 6 5 4 3 2 1 E Gsdmd / OI 1 NAG Lipo Extracellular space OI 3 OI 1 OI 3 IL1β (pg/ml) 1 8 6 4 2 Gsdmd / OI 1 OI 3 Gsdmd / OI 1 OI 3 p17 IL1β 1 9 8 7 6 5 4 3 2 1 Gsdmd / NAG alone F Lipo/NAG Lysate NAG Lipo/NAG UT IL1β (pg/ml) 3 2 1 Gsdmd / Gsdmd / NAG alone Lipo/NAG NAG Lipo/NAG NAG Lipo/NAG NAG Lipo/NAG p17 IL1β βactin **** G 1 9 8 7 6 5 4 3 2 1 Gsdmd / POVPC PGPC POVPC PGPC H IL1β (pg/ml) 15 1 5 Gsdmd / POVPC PGPC POVPC PGPC I J K L 1 9 8 7 6 5 4 3 2 1 Gsdmd / 5 m K 5 m K OI 3 OI 3 5 m K OI 3 OI 3 5 m K IL1β (pg/ml) 1 8 6 4 2 Gsdmd / 5 m K 5 m K OI 3 OI 3 5 m K OI 3 OI 3 5 m K 1 9 8 7 6 5 4 3 2 1 Gsdmd / 5 m K POVPC PGPC POVPC POVPC 5 m K PGPC PGPC 5 m K IL1β (pg/ml) 15 1 5 Gsdmd / 5 m K POVPC PGPC POVPC POVPC 5 m K PGPC PGPC 5 m K Figure 2

Figure 2: Role of GDD and potassium in macrophage hyperactivation. Related to Figure 2. (A) LDH cytotoxicity measurements of cellfree culture supernatants. and Gsdmd/ clone 2 ibds were primed with for 4 hours (or not), and then infected with A113 ΔoatA at an OI of 1 and 3. (B) IL1β ELIA measurements of cellfree culture supernatants. and Gsdmd/ clone 2 ibds were primed with for 4 hours (or not), and then infected with A113 ΔoatA at an OI of 1 and 3. (C) LDH cytotoxicity measurements of cellfree culture supernatants. and Gsdmd/ clone 1 ibds were primed with for 4 hours (or not), and then transfected with NAG complexed with lipofectamine 2 or treated with NAG alone for 6 hours. (D) IL1β ELIA measurements of cellfree culture supernatants. and Gsdmd/ ibds clone 1 were primed with for 4 hours (or not), and then transfected with NAG complexed with lipofectamine 2 or treated with NAG alone for 6 hours. **** p<.1 as determined by twoway ANOVA with Tukey s multicomparison correction. (E) Immunoblot analysis of cleaved IL1β in cellfree culture supernatants from and Gsdmd / ibds after 4 hours of priming (or not), and then transfected with NAG complexed with lipofectamine 2 or treated with NAG alone for 6 hours. (F) Immunoblot analysis of cellassociated cleaved IL1β in Gsdmd/ clone 1 ibds after 4 hours of priming (or not), and then transfected with NAG complexed with lipofectamine 2 or treated with NAG alone for 6 hours. (G) LDH cytotoxicity measurements of cellfree culture supernatants. and Gsdmd/ clone 2 ibds were primed with for 4 hours (or not), and then treated with PGPC or POVPC for 6 hours. (H) IL1β ELIA measurements of cellfree culture supernatants. and Gsdmd/ clone 2 ibds were primed with for 4 hours (or not), and then treated with PGPC or POVPC for 6 hours.

(I) LDH cytotoxicity measurements of cellfree culture supernatants. and Gsdmd/ clone 1 ibds were primed with for 4 hours (or not), and then infected with A113 ΔoatA at an OI of 3. timulations contained an additional concentration of m K or 5 m K. (J) IL1β ELIA measurements of cellfree culture supernatants. and Gsdmd/ clone 1 ibds were primed with for 4 hours (or not), and then infected with A113 ΔoatA at an OI of 3. timulations contained an additional concentration of m K or 5 m K. (K) LDH cytotoxicity measurements of cellfree culture supernatants. and Gsdmd/ clone 1 ibds were primed with for 4 hours (or not), and then treated with PGPC or POVPC for 6 hours. timulations contained an additional concentration of m K or 5 m K. (L) IL1β ELIA measurements of cellfree culture supernatants. and Gsdmd/ clone 2 ibds were primed with for 4 hours (or not), and then treated with PGPC or POVPC for 6 hours. timulations contained an additional concentration of m K or 5 m K. Data with error bars are represented as mean ± E. Each panel is a representative experiment of at least 3 repeats.

ul tim ns at ed PO VP C PG PC L P on l PO y VP C PG PC U IL1β (pg/ml) 25 on O I3 ly O I1 1 12 O I3 PG N 5 µg l /m ly l /m on µg 5 at ed IL1β (pg/ml) 2 ul tim ns PG N U ns tim ul a AT ted P N ig er 5 m ic in 1 µ L P on AT ly P N ig er 5 m ic in 1 µ U IL1β (pg/ml) 1 at ed IL1β (pg/ml) 12 O I1 ul tim ns U A E ACcitrine BDs 8 6 4 2 B ACcitrine BDs 15 1 5 C ACcitrine BDs 8 6 4 2 ATP Nigericin PGN OI 3 A113 ΔoatA POVPC PGPC D 2 ACcitrine BDs 15 1 5 Figure 3

Figure 3: IL1β release and bright field morphology of ACcitrine macrophages treated with pyroptotic and hyperactivating stimuli. Related to Figure 3. (A) IL1β ELIA measurements of cellfree culture supernatants. ACcitrine BDs were primed with for 3 hours (or not), and then treated with 5 m ATP or nigericin for 2 hours. (B) IL1β ELIA measurements of cellfree culture supernatants. ACcitrine BDs were primed with for 3 hours (or not), and then treated with 5 µg/ml of extracellular PGN from. aureus for 6 hours. (C) IL1β ELIA measurements of cellfree culture supernatants. ACcitrine BDs were primed with for 3 hours (or not), and then treated with A113 ΔoatA at an OI of 1 and 3 for 12 hours. (D) IL1β ELIA measurements of cellfree culture supernatants. ACcitrine BDs were primed with for 3 hours (or not), and then treated with POVPC or PGPC for 6 hours. (E) Bright field imaging of live cell ACcitrine expressing BDs primed with for 3 hours with second stimulations of 5 m ATP, nigericin, 5 µg/ml PGN from. aureus, OI 3 of A113 ΔoatA, POVPC or PGPC for 1622 hours. Data with error bars are represented as mean ± E. Each panel is a representative experiment of at least 3 repeats.

A hr 7 hr CA FCA EGFP fusion construct: GDD GDD (I15N) CTGDD NTGDD NTGDD (I15N) B hr 7 hr Count 7AAD EGFP fusion construct: GDD GDD (I15N) CTGDD NTGDD NTGDD (I15N) C upernatant FITC Flourescence (% aximum) 1 9 8 7 6 5 4 3 2 1 4 kda Dextran Caspase11 / GDD 2 kda Dextran Caspase11 / GDD 2 kda Dextran Caspase11 / GDD 4 kda Dextran Caspase11 2 kda Dextran Caspase11 2 kda Dextran Caspase11 2 4 6 8 1 12 Time (min) D 1 9 8 7 6 5 4 3 2 1 Pellet upernatant C11 / GDD 3 min C11 / GDD 12 min C11 3 min C11 12 min GDD 3 min GDD 12 min Figure 4

Figure 4: Flow cytometry analysis of 293T cells expressing GDD variants and release of biomolecules from PC:P liposomes. Related to Figure 4. (A) Forward and side scatter gating to exclude cell debris from analysis of and 7 hour time points postelectroporation with GDD fusion constructs. (B) Representative 7AAD positivity gating on cells that reside in the gating from (A) from hour and 7 hour time points postelectroporation with GDD fusion constructs. (C) Plate reader fluorescence quantification of fluorescent dextran release from unextruded PC:P liposomes loaded with 4, 2, and 2 kda W treated with caspase11, GDD, or cotreatment for indicated times. Data are represented as mean ± E of technical replicates from one representative experiment of 3 repeats. (D) Plate reader colorimetric quantification of tetrameric LDH enzymatic activity release from PC:P liposomes loaded with LLDH isolated from rabbit muscle treated with caspase11, GDD, or cotreatment for indicated times. Data are represented as mean ± E of combined technical replicate means from 3 independent repeats.

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