Afterwards, cells were allowed to expand for further analyses and injections

uce ATF4 expression and screened by CP 868596 web SDS-PAGE/immunoblot analysis. To detect the presence of out-of-frame insertions/deletions in all ATF4 alleles, the genotype of knock-out clones was verified by Sanger DNA sequencing. To delete the entire 70 kb corresponding to NLRP1 gene locus, HeLa, THP-1 and K562 cells were simultaneously co-transfected with 2 different CRISPR-Cas9 plasmids targeting the regions in proximity of the start codon and of the stop codon respectively, together with an EGFP plasmid to allow single cells sorting. After 2 weeks, clones were screened by genomic PCR. All NLRP1-/- clones were identified by the presence of PCR amplification using a forward primer in the 5’UTR and a reverse primer in the 3’UTR and by the contemporary absence of amplification products in each of the 5’UTR and the 3’UTR regions. Locus deletion was verified by Sanger DNA sequencing and mRNA expression was analyzed by RT-PCR/qPCR. All gene knock-out clones are available upon request. Results NLRP1 is up-regulated during ER stress conditions Since ER stress was shown previously to activate the NLRP3 inflammasome, we explored the effects of ER stress on NLRP1 gene expression. Stimulation of the PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19740312 human monocytic cell line THP-1 with two different ER stress inducers, tunicamycin –to inhibit Nlinked glycosylation–and thapsigargin –to inhibit the ATP-dependent calcium pump SERCA-, produced marked increases in NLRP1 mRNA expression. In contrast, the well-known NLRP3 inflammasome activator uric acid did not. We further tested ER stress-dependent NLRP1 induction in Jurkat cells, a human T cell line that normally does not express NLRP3 but has elevated NLRP1 mRNA basal levels. The ER-Golgi transport blocker brefeldin A and TG both induced time-dependent increases in NLRP1 mRNA levels in Jurkat cells, while the TLR7 ligand and NLRP3 inflammasome activator R837 had no effect on NLRP1 mRNA expression. Similarly, time-dependent NLRP1 up-regulation was observed in HeLa epithelial cancer cells that have very low NLRP1 mRNA basal levels. Time-course experiments with either BFA or TG showed that ER stress increased NLRP1 expression after 12 or 6 hours respectively. To test whether ER stress specifically induces NLRP1 gene expression, we stimulated HCT116 human colon cancer cells with various inflammatory stimuli and measured mRNA expression of both NLRP1 and NOD1, which is another human NLR family member. Induction of NLRP1 but not NOD1 gene expression was observed only during ER stress conditions induced by BFA treatment but not by other pro-inflammatory stimuli. Next, to perform a more comprehensive analysis, we stimulated HeLa cells with TM for different times and performed a transcriptome study by total RNA-sequencing. We focused on all currently known human NLR genes and found that among the 17 NLRs only NLRP1 and NLRC5 were expressed at any time point, while NLRP1 alone showed up-regulation under ER stress conditions. We PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19740489 next investigated whether increased NLRP1 mRNA levels upon ER stress correlate with increased NLRP1 protein expression. Immunoblot analysis of cell extracts from BFA-treated HeLa, THP-1 and K562 cells showed up-regulation of a 130 kDa band, presumably corresponding to NLRP1 N-terminal autocleaved fragment. Since a barely visible 130 kDa 5 / 16 ATF4 Controls NLRP1 Expression during ER Stress Fig 1. NLRP1 mRNA and protein are up-regulated upon ER stress. Un-differentiated THP-1 cells were treated with the indicated stimuli for 6 hours. NLRP1 lev