Ne per nuclear and cytoplasmic extraction reagent

The nuclear and cytoplasmic fractions were extracted using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific). Total RNA from whole-cell lysates or the nuclear and cytoplasmic fractions were isolated using TRIzol (Life Technologies, Carlsbad, CA). For RNase R treatment, 2 μg of total RNA was incubated 20 min at 37 °C with or without 3 U μg⁻¹ of RNase R (Epicentre Technologies, Madison, WI), and the resulting RNA was subsequently purified using an RNeasy MinElute cleaning Kit (Qiagen). To quantify the amount of mature miRNA, we used TaqMan MicroRNA assays (Life Technologies) and small nuclear U6B (RNU6B) RNA as an internal standard. To quantify the amount of mRNA and circRNA, cDNA was synthesized with the PrimeScript RT Master Mix (Takara, Dalian, China) from 500 ng of RNA. The real-time PCR analyses were performed using SYBR Premix Ex Taq II (Takara). In particular, the divergent primers annealing at the distal ends of circRNA were used to determine the abundance of circRNA. To determine the absolute quantity of RNA, the purified PCR product amplified from cDNA corresponding to the circHIPK3 sequence was serially diluted to generate a standard curve. The primers are listed in Supplementary Table 3.

Stimulated cells and tissue samples were harvested for protein extraction and western blotting analysis with standard protocols as previously described^{12 (link),56 (link)}. Cytoplasmic fractionations were performed with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific, Rockford, IL, USA) according to the manufacturer’ s protocol. Briefly, cells were harvested, washed and pelleted. Then added ice-cold CER I to the cell pellet and incubated the tube for 10 min on the ice. After that, added ice-cold CER II to the tube and incubated for 1 min on the ice. The volume ratios of cell pellets, CER I and CER II are 10: 100: 5.5. At last, centrifuged the tube for 5 min at 16, 000 × g and transferred the supernatant to a clean pre-chilled tube. Stored this tube in −80 °C until use. The following antibodies were used: anti-GAPDH (1:10000; Genetex, Irvine, CA, USA); anti-DAPK1, anti-MLC (1:1000; Abcam, Cambridge, MA, USA); anti-p-MLC (1:500; Cell Signaling Technology, MA, USA); anti-IL-1β (1: 2000; R&D Systems, Wiesbaden, Germany); anti-NLRP3, anti-caspase-1, anti-ASC (1:1000; Adipogen, San Diego, CA, USA)]; anti-cathepsin B (1:300; Santa Cruz Biotechnology, Santa Cruz, CA, USA); secondary antibodies (1:400; Jackson ImmunoResearch Laboratories, PA, USA). The intensity of protein bands was analyzed with the Image J software (NIH) and normalized to GAPDH.

Proteins from PBMCs were isolated with the NucleoSpin RNA/Protein Kit (Macherey-Nagel). Nuclear and cytoplasmic proteins from transfected HEK293T cells were obtained with the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific). Concentrations were measured with Protein Quantification Kit (Macherey-Nagel).