Gasdermin A and Gasdermin B localize the cytoskeleton in keratinocyte.

(A) N/TERT-1, (B) HaCaT and (C) Colo205 cells were followed by subcellular fractionation into cytoplasmic, membrane, nuclear, chromatin bound, cytoskeleton as described in Methods. Each fraction was validated by immunoblotting with antibodies against LDHA, E-cadherin, Lamin A/C, DSG1, and KRT19. Numbers below the blots indicate individual purified fraction sample. Whole cell lysates were immunoblotted with the indicated antibodies. Molecular weights (MW, kDa) are represented on the left.

Gasdermin C localizes to the cell membrane and is not cleaved by caspase 8.

(A) N/TERT-1, (B) HaCaT and (C) Colo205 cells were followed by subcellular fractionation into cytoplasmic, membrane, nuclear, chromatin bound, cytoskeleton as described in Method. Each fraction was validated by immunoblotting with antibodies against LDHA, E-cadherin, Lamin A/C, DSG1, and KRT19. Numbers below the blots indicate individual purified fraction sample. (D) N/TERT-1 cells were pretreated with cycloheximide (C, 100 µg/ml) for 30 minutes, followed by TNF (T, 100 ng/ml) treatment for another 24 hours. * indicates cleavage form of gasdermin D. # indicates cleavage form of gasdermin E. (E) Hela cells were pretreated with cycloheximide (C, 100 µg/ml), and BV6 (B, 10 µM) for 30 minutes, followed by TNF (T, 100 ng/ml), and staurosporine (STS, 10 µM) treatment for another 4 hours respectively. * indicates cleavage form of gasdermin D. # indicates cleavage form of gasdermin E. (A-E) Whole cell lysates were immunoblotted with the indicated antibodies. Molecular weights (MW, kDa) are represented on the left. (D, E) Equal protein loading was confirmed by Ponceau S staining.

Calyculin A triggers translocation of GSDMA and GSDMB from cytoskeleton to plasma membrane.

(A) N/TERT-1, (E) Colo205 cells were followed by subcellular fractionation into cytoplasmic, membrane, nuclear, chromatin bound, cytoskeleton as described in Method. Each fraction was validated by immunoblotting with antibodies against LDHA, β-tubulin, E-cadherin, Lamin A/C, Vimentin, and pan-KRT. Numbers below the blots indicate individual purified fraction sample. (A, E) Whole cell lysates were immunoblotted with the indicated antibodies. Molecular weights (MW, kDa) are represented on the left. (B) N/TERT-1 cells were treated with calyculin A (20nM) for 12 hours and stained with Yoyo-1 dye. Representative images from Incucyte live-cell imaging are shown. Red arrow indicates cell membrane ballooning. (C) N/TERT-1 cells were pretreated with Disulfiram (5 µM) for 30 minutes, followed by calyculin A (20 nM) treatment. (D) N/TERT-1 cells were transfected with either siMock or siGsdmaA/B and subsequently treated with calyculin A (20 nM). Cell death was quantified by Yoyo-1 dye uptake using Incucyte live-cell imaging. Graph represents percentage of cell death. Data are presented as mean ± SEM.

Endocytosis is required for gasdermin A and gasdermin B-dependent pyroptosis.

(A) N/TERT-1 cells were pretreated with Pitstop2 (25 µM) for 30 minutes, followed by calyculin A (20 nM) treatment for 12 hours. Representative images from Incucyte live-cell imaging are shown after Yoyo-1 dye staining. (B, C) N/TERT-1 cells were pretreated with (B) Dynasore (100 µM) or (C) Pitstop2 at indicated concentration for 30 minutes, followed by calyculin A (20 nM) treatment. Cell death was quantified by Yoyo-1 dye uptake using Incucyte live-cell imaging. Graphs represent percentage of cell death. Data are presented as mean ± SEM.

Calyculin A acantholysis releases post-translationally modified keratin.

(A, B) N/TERT-1 cells were pretreated with Dynasore (100 µM) for 30 minutes, (C) N/TERT-1 cells were transfected with either siMock or siGsdmaA/B followed by calyculin A (20 nM) treatment for the indicated times. Cell lysates and supernatants separated as described in Methods. Whole cell lysates and supernatants were immunoblotted with the indicated antibodies. Molecular weights (MW, kDa) are represented on the left. Equal protein loading was confirmed by (A) GAPDH, (B) Ponceau S staining or (C) LDHA and Actin.