LRP1 inflammasome in response to ER perturbations. We found that both PERK and IRE1 stimulate NLRP1 gene transcription through the transcription factor ATF4, involving a mechanism that is independent of XBP-1 mRNA splicing. Materials and Methods Cell culture Cell lines were cultured at 37C with 5% CO2. THP-1, K562 and Jurkat cells were maintained in RPMI-1640 medium, HeLa cells in Dulbecco’s modified Eagle’s medium and HCT116 cells in McCoy’s-5A medium. All media were supplemented with 10% heat-inactivated fetal bovine serum PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/1974422 and penicillin-streptomycin. All cell lines were obtained from ATCC. Reagents, plasmids, and antibodies DMSO, tunicamycin, thapsigargin, brefeldin A, actinomycin D, DRB and Forskolin were purchased from Sigma. Monosodium urate crystals 
were prepared from uric acid as previously described. PolyI:C, flagellin, MDP and PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19744306 R837 were from Invivogen. All siRNA were purchased from Ambion. Different shRNA were cloned in pLKO.1 and/or in Tet-pLKOpuro vectors as previously described. A complete list of siRNA and shRNA used is presented in S1 2 / 16 ATF4 Controls NLRP1 Expression during ER Stress Cell Signaling Technology, ATF6 antibody was from Abcam, and XBP-1s antibody was from Biolegend. ATF4 antibody for ChIP was from Santa Cruz. NLRP1 antibody was from R&D Systems. RNA extraction, semiquantitative RT-PCR and real-time qPCR After ER stress induction, cells were washed twice with PBS and total RNA extraction was performed using an RNeasy kit. RNA concentration and purity was measured using a NanoDrop spectrophotometer. A total of 2g RNA was retro-transcribed using SuperScript III reverse transcriptase and used for subsequent PCR reaction. Platinum TAQ was used for semi-quantitative RT-PCR. Real time qPCR was performed using TaqMan universal master mix II and different TaqMan assays. A complete list of primers and assays for RT-PCR and qPCR is provided in S2 Adenovirus, lentiviruses and cell transduction Murine Xbp-1s and murine Atf4 adenoviruses used here were previously described. The shRNA lentivirus plasmids were packaged by transfection into 293T cells, as already described. Stably transduced cells were selected in media containing puromycin. Conditional shRNA were selected and maintained in media containing tetracycline-free serum. Luciferase vectors and assays The putative NLRP1 promoter region 2555bp GW 501516 upstream of the ATG codon, including 555 nucleotides corresponding to the 50 untranslated region was cloned from human genomic DNA into the pGL4.12 luciferase vector by PCR using the KpnI and SacI restriction sites. Four shorter constructs were cloned using internal primers. All plasmid cloning was verified by DNA sequencing. Luciferase vectors, together with the renilla plasmid pGL4.74 were transiently co-transfected into HeLa cells using JetPrime according to the manufacturer’s protocol. After 24 hours, cells were treated with 2M BFA overnight and luciferase signals were detected using the Dual-luciferase reporter assay system. Cell transfection, immunoprecipitation and immunoblot analysis HeLa cells were seeded into 6-well plates at a density of 1 x 106 cells per well. Two different siRNAs were transfected using RNAiMAX reagent according to manufacturer’s protocol. After 24 hours, cells were treated overnight with 2M BFA to induce ER stress. The following day the cells were washed twice with PBS and lysed in 1x SDSsample buffer SDS, 0.01% phenol red, 10% glycerol and 20mM DTT). Endogenous NLRP1 protein was pulled-down i