Proteinase k

Approximately 1 × 10⁷ BMDMs were prepared and activated as described.
The SimpleChIP^® Enzymatic Chromatin IP Kit (Cell Signaling Technology) was used according to the manufacturer's instructions. Samples were subjected to immunoprecipitation using either rabbit anti-H3K9me3 antibody or a control IgG antibody. Fragmented DNAs were isolated from histones by Proteinase K and purified using spin columns (both from Cell Signaling Technology). DNA was used as a template for qPCR using the indicated primer sets spanning the tnf-α promoter (Supplementary Table S3). Fold enrichments were normalized and calculated based on the total amount of 2% input and presented as a relative quantifications using the 2^−∆∆ct method.

ChIP assays were performed using the Simple ChIP Enzymatic Chromatin IP Kit (Agarose Beads), in accordance with the manufacturer’s protocol (Cell Signaling Technology, Danvers, MA, USA). PC3 cells seeded in 10 cm dishes were transfected with siRNA for 24 h. Cells were cross-linked with 1% formaldehyde for 10 min at room temperature and stopped by addition of 0.2 M glycine. Chromatin was digested with micrococcal nuclease to an average size of 150 bp; the reaction was stopped by addition of 0.5 M EDTA. Nuclear membranes were lysed by sonication with 8 sets of 10 s pulses, using a Diagenode Bioruptor (Diagenode SPA, Región de Valparaiso, Chile). After centrifugation had been performed, anti-HOXB13 (Cat# PA5-78327; Thermo Fisher Scientific), or Normal Rabbit IgG (Cat# 2729; Cell Signaling Technology) antibodies were added to lysates; the mixtures were rotated at 4 °C overnight. Protein beads were then added, and the lysates were rotated for 2 h. Beads were then washed, and DNA was eluted and treated with proteinase K (Cell Signaling Technology). DNA was purified with spin columns and used as template for qPCR. Fold enrichment relative to input levels was quantified by qPCR using the SYBR Green PCR master mix and 7300 Real-Time PCR system. The RPL30 locus was used as the positive control. Primer sequences for ChIP qPCR are listed in Table S3.

The SimpleChIP Plus Sonication Chromatin IP Kit (56383, Cell Signalling) was used, following manufacturer’s guidelines. In brief, after resuspension of the chromatin pellet in ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8.0 plus 1 X protease inhibitor cocktail), samples were subjected to fragmentation using a Bioruptor pico sonicator (Diagenode) for 5 cycles (30 s ON and 30 s OFF). 5 μg of sonicated, cross-linked chromatin sample per condition was diluted in a 1:4 ratio in 1X ChIP buffer (Cell Signalling) plus 1X protease inhibitor cocktail (Cell Signalling) and subjected to immunoprecipitation with 2.5 μg H3K27ac antibody (Abcam) overnight at 4 °C with rotation. 30 μl of ChIP-Grade Protein G Magnetic Beads (Cell Signalling) were then added to each IP reaction and incubated for 2 h at 4 °C with rotation, prior to a series of low and high salt washes and elution of antibody-protein-DNA complexes in 1X ChIP Elution Buffer. Enriched chromatin samples were then incubated at 65 °C for 2 h with 40 μg Proteinase K (Cell Signalling) to reverse cross-links prior to purification of DNA using the DNA Purification Kit (Cell Signalling) as per manufacturer’s guidelines. DNA was eluted in 50 μL of elution buffer prior to subsequent qPCR using the Luna Universal qPCR Master Mix kit (New England BioLabs).

ChIP assay coupled with quantitative PCR (ChIP-qPCR) was conducted to confirm the DNA-binding ability of Foxo1, HDAC2, or H3K9AC protein with GSDMD gene using a Simple ChIP Plus Enzymatic Chromatin IP Kit (CST) according to the manufacturer's protocol. The H9C2 cells were treated with protease inhibitor cocktail coupled with gradually decreased formaldehyde (37% ~1.5%) for 20 min and then blocked by glycine solution for 5 min at room temperature. Cross-linked chromatins were broken into DNA fragments using DNA micrococcal nuclease at 37°C for 20 min. ChIP reaction was conducted using 5 μg of antibody against Foxo1 (1 : 30, ab39670, Abcam), H3K9AC (ab32117, 20 μg/ml), HDAC2 (1 : 60, ab32117), or 1-2 μl IgG (negative control) enriched in protein G magnetic beads (Invitrogen) at 4°C overnight. The immunoprecipitated complexes were then eluted using ChIP elution buffer at 65°C for 30 min, and DNA were decross-linked from proteins using proteinase K (Cell Signaling Technology) at 65°C for 2 h. The products were used for PCR of GSDMD gene using the primer pairs listed in Table 1. PrimeSTAR® HS DNA Polymerase (Takara) and a SYBR Green qPCR SuperMix kit (DBI Bioscience, Shanghai, China) were employed for PCR amplification. The DNA samples obtained were purified and subjected to PCR analysis and 2% agarose gel (Biowest, Nuaillé, France). All the reactions were performed in triplicate.