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ASC knockout or knockdown in THP-1 cells

Canonical inflammasome signaling in THP1-KO-ASC cells

ASC (apoptosis-associated speck-like protein containing a CARD domain, also known as PYCARD) is a protein adaptor important in canonical inflammasome responses [1]. ASC's bipartite composition, consisting of one PYD and one CARD domain, allows the recruitment of the CARD-containing pro-caspase-1 to canonical inflammasome sensors that do not contain a CARD domain, such as NLRP3, AIM2, and Pyrin [1].

More details

To foster research on the ASC adaptor, InvivoGen provides cellular tools that have been generated from the human monocytic THP1 cell line through either a stable knockout or knockdown of the ASC gene. THP-1 cells are widely used for inflammasome studies due to their high expression levels of NLRP3, ASC, and pro-caspase-1.

• THP1-KO-ASC cells – Knockout (KO) of the ASC gene

• THP1-defASC cells – Knockdown (KD) of the ASC gene

These cell lines exhibit a similar phenotype but have dramatically different genotypes. They share the common phenotype of abrogation of mature IL-1β secretion. However, THP1- defASC cells exhibit a knockdown of ASC gene expression whereas THP1-KO-ASC cells possess a KO of the ASC gene. Indeed, THP1- defASC cells have a maximum 3-fold reduction in ASC expression (RT-qPCR), whereas THP1-KO-ASC cells have no ASC protein expression (Western blot). These cell lines are useful tools in the study of the ASC-dependent inflammasome responses and can also be used as control cell lines for the screening of novel therapeutics that target the ASC signaling pathways.

Features of THP1-KO-ASC cells:

Generated from the parental cell line THP1-Null2
Verified biallelic KO of the ASC gene (sequencing, PCR, and Western blot)
Complete abrogation of mature IL-1β secretion upon activation of the canonical or non-canonical inflammasomes

Features of THP1-defASC cells:

Verified KD of the ASC gene (RT-qPCR)
Significantly reduced mature IL-1β secretion upon activation of the canonical inflammasome
Highly referenced in inflammasome-related literature (see citations)

InvivoGen also provides THP1-ASC-GFP reporter cells that express an inducible ASC::GFP fusion protein to monitor the priming and the activation steps of ASC-dependent inflammasomes using time-lapse confocal or high-resolution fluorescence microscopy.

For detecting and quantifying the release of mature human (h)IL-1β, InvivoGen provides HEK-Blue™ IL-1β sensor cells, which express an NF-κB-inducible SEAP reporter gene. QUANTI-Blue™ Solution allows rapid colorimetric detection and measure of SEAP activity by reading the optical density at 630-650 nm.

For detecting and quantifying pyroptotic cell death, InvivoGen provides THP1-HMGB1-Lucia ™ cells, which express a cytoplasmic HMGB1::Lucia luciferase fusion protein that is released in the supernatant upon pyroptosis. The activity of the Lucia luciferase reporter protein can be readily assessed using the QUANTI-Luc ™ detection reagent.

Download our Practical guide on Inflammasomes

Reference:

1. Mathur A. et al., 2017. Molecular mechanisms of inflammasome signaling. J. Leuk. Biol. 103:233.

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