biostudies-arrayexpressYing Shiang LimHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15754Inflammasomes are multicomplex proteins which drive pro-inflammatory cytokine responses and pyroptosis in innate immunity. Of which, the NLRP3 inflammasome is the most extensively studied, especially in myeloid immune cells such as macrophages. Conversely, the regulation of NLRP3 in non-immune cell types such as keratinocytes remains elusive. To understand this under specific immune environments, immortalized N/TERT keratinocytes were stimulated overnight with or without 50ng/ml IFNγ (technical triplicates per group ,Untreated (UNT) vs stimulated (IFNy), 6 samples in total). Total RNA was extracted from the cells by TRIzol-based RNA extraction for quantification and quality check, library construction and RNA-seq (paired-end sequencing) was performed according to standard protocols from Macrogen (South Korea) on the NovaSeq 6000 platform.biostudies-arrayexpressNucleic Acid Extraction - Total RNA extraction was performed using TRIzol reagent. Isopropyl alcohol is added to the the cells homogenized in TRIzol, followed by centrifugation.Sample Treatment - Immortalized N/TERT keratinocytes are seeded on a 6-well plate and treated at 80% confluency with 50ng/ml of IFN-gamma (or untreated) overnight.Sample Collection - TRIzol is added directly to the cells in a 6-well plate after overnight stimulation.Sequencing - The sequencing libraries were multiplexed and loaded on the flowcell on the Illumina NovaSeq 6000 instrument according to manufacturer’s instructions. The samples were sequenced using a Pair-End (PE) configuration.Library Construction - Library construction was conducted via standard protocol with Macrogen (South Korea).OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Sequencing reads were mapped to the reference genome (Homo sapiens, GRCh38) using HISAT2. Transcripts were assembled from the aligned reads using StringTie, and expression profiles were quantified as read counts and normalized values, represented as transcripts per kilobase million (TPM) for each sample, taking into account transcript length and coverage depthMetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000<h4>ABSTRACT</h4> While NLRP3 has been extensively studied in myeloid cells, its existence and regulation in epithelial cells, including keratinocytes, are unclear. In fact, whether human keratinocytes express a functional NLRP3 inflammasome at all remains a matter of debate in the inflammasome field. Here, we provide additional evidence that NLRP3 is repressed in human keratinocytes cultured under non-inflammatory conditions but can be sharply induced by interferon-γ (IFNγ)—but not lipopolysaccharide (LPS). In this IFNγ-primed state, not all established NLRP3 activators are specific to NLRP3. We report that nigericin-driven keratinocyte pyroptosis occurs via both NLRP1 and NLRP3, whereas Staphylococcus aureus α-hemolysin (Hla) exclusively and nonredundantly activates NLRP3, even though both require K+ efflux. Furthermore, in the presence of T cells, certain virulent S. aureus strains can cause NLRP3-dependent pyroptotic death in keratinocytes in vitro through the cooperative actions of superantigens (SAgs) and Hla. In summary, our findings establish the strict inducibility and functional relevance of the NLRP3 inflammasome in non-myeloid, epithelial cells in vitro. These results resolve conflicting reports and position keratinocytes as a context-specific, non-hematopoietic cellular model for studying NLRP3 activation in host-microbe interactions at barrier tissues. <h4>KEY FINDINGS</h4> Additional evidence that NLRP3 is absent in resting, nonstimulated human keratinocytes in vitro IFNγ, but not LPS, is a potent ‘priming’ signal for NLRP3 in human keratinocytes in vitro In IFNγ-primed keratinocytes, S. aureus α-hemolysin (Hla) selectively activates NLRP3, whereas nigericin activates both NLRP1 and NLRP3 in vitro SAg and Hla kill keratinocytes via NLRP3-driven pyroptosis in the presence of T cells in vitro <h4>GRAPHICAL ABSTRACT</h4>RNA-seq of coding RNAHomo sapiensClarifying the priming requirement and activator specificity for the NLRP3 inflammasome in human keratinocytes in vitroIFNγ priming enables NLRP3 inflammasome activation in human keratinocytes in vitroPritisha Rozario, Ying Shiang Lim, Shirley Suet Lee Ding, Muhammad Jasrie Firdaus, Stephen Wearne, Wong Han Siang Brandon, Rae Chua, Kim S Robinson, Julie Tung Sem Chu, Liu Meng, Shan Sophie Carrie CAI, Sern Ting Eugene TAN, Soon Keong Wee, Mart Matthias Lamers, Navin Kumar Verma, Xia Yun, Eric Peng Huat Yap, John E. A Common, Franklin ZhongFranklin ZhongYing Shiang LimfalseBulk RNA-seq of immortalized N/TERT keratinocytes stimulated with IFNγInflammasomes are multicomplex proteins which drive pro-inflammatory cytokine responses and pyroptosis in innate immunity. Of which, the NLRP3 inflammasome is the most extensively studied, especially in myeloid immune cells such as macrophages. Conversely, the regulation of NLRP3 in non-immune cell types such as keratinocytes remains elusive. To understand this under specific immune environments, immortalized N/TERT keratinocytes were stimulated overnight with or without 50ng/ml IFNγ (technical triplicates per group ,Untreated (UNT) vs stimulated (IFNy), 6 samples in total). Total RNA was extracted from the cells by TRIzol-based RNA extraction for quantification and quality check, library construction and RNA-seq (paired-end sequencing) was performed according to standard protocols from Macrogen (South Korea) on the NovaSeq 6000 platform.2025-10-30T00:00:00Z2026-05-27T17:00:25.054Z2025-10-16T11:25:47.698ZE-MTAB-15754ERP182296EFO_0002944EFO_0004170EFO_0005518EFO_0003816EFO_0003738EFO_0004184EFO_000396910.1101/2025.08.13.669639