b c * a Supplementary Figure 1. NLRP1 knockdown suppresses tumor growth in vivo. (a) 1205Lu tumors with NLRP1 shRNA from Santa Cruz Biotechnology. (b) 1205Lu tumors with NLRP1 shRNA from Origene. (c) HS294T tumors with NLRP1 shRNA from Santa Cruz Biotechnology. Mean ± s.e.m., n = 10 (a), 6 (b) or 8 (c). *P < 0.05.

1205Lu HS294T Supplementary Figure 2. NLRP1 knockdown increases apoptosis analyzed by flow cytometry. Cells were exposed to 1 μM AMD for 18 h. (a) Control shRNA-transduced cells treated with DMSO as vehicle. (b) NLRP1 shRNA-transduced cells with DMSO. (c) Control shRNA-transduced cells with AMD. (d) NLRP1 shRNA-transduced cells with AMD. a b c 0.22 3.62 /1.69/1 1.1 1.69/1.41 d Annexin V PI

THP-1 Supplementary Figure 3. Annexin V staining shows that NLRP1 knockdown by NLRP1 siRNA had no effects on apoptosis in THP-1 cells in the absence or presence of AMD. Mean ± s.e.m., n = 3. KD, knockdown.

IgG NLRP1 1205Lu NLRP3 IP HS294T Heavy chain Light chain 150 kDa 100 kDa 37 kDa 50 kDa 75 kDa 25 kDa 20 kDa 15 kDa 250 kDa M Supplementary Figure 4. Ponceau S (Sigma) staining of the blot depicted in Figure 5a shows the ~55 kDa heavy chains and ~23 kDa light chains of the immunoprecipitating antibody. M, molecular weight marker.

WM35 WM115 * *** * ** Supplementary Figure 5. NLRP1 knockdown enhances caspase-3/7 activity in WM35 and WM115 treated with DMSO as vehicle, AMD or CPT. Mean ± s.e.m., n = 3. *P < 0.05, **P < 0.01, and ***P < 0.001. KD, knockdown.

*** * 1205Lu *** * HS294T Supplementary Figure 6. NLRP1 knockdown results in decreased caspase-1 activity in 1205Lu and HS294T cells treated with AMD for 18 h. *P < 0.05 and ***P < 0.001. Mean ± s.e.m., n = 3. KD, knockdown.

1205Lu HS294T Supplementary Figure 7. NLRP1 inflammasome activation by LT reduces apoptosis assayed by flow cytometry. Cells were stimulated with LT for 2 h, then AMD for another 18 h. (a) Vehicle. (b) LT. (c) AMD. (d) LT plus AMD. a b c 0.22 3.62 /1.69/1 1.1 1.69/1.41 d Annexin V PI

THP-1 ** *** * 1205Lu ** * Supplementary Figure 8. LT enhances caspase-1 activity in differentiated THP-1, but not in 1205Lu cells, treated with LT for 2 h and then with AMD or CPT for 18 h. MSU and IL-1α were used as controls. Mean ± s.e.m., n = 3. *P < 0.05, **P < 0.01, and ***P < 0.001.

Caspase-2 Caspase-1 Caspase-9 CyPA HSP90 Lamin B Untreated LT PMA PMA + LT THP-1 Cytoplasmic Untreaed Nuclear 1205Lu Supplementary Figure 9. Cytoplasmic localization of caspase-2 and -9 in THP-1 and 125Lu cells assayed by Western blot. For THP-1 cells, the subcellular fractions were from Figure 1d. CyPA, cyclophilin A. 20 μm