ASC Knockout & Knockdown Monocytes

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.

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Reference:

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

Figures

Lysates from THP1-Null2 (WT) and THP1-KO-ASC (KO) cells were analyzed using an anti-human ASC antibody, followed by an HRP‑conjugated anti-rabbit secondary antibody. The arrow indicates the expected band for the ASC protein (27 kDa).

~3x10⁵ THP1-Null2  (blue) and THP1‑KO-ASC cells (red) were incubated for 3h at 37°C with LPS-EK (1 μg/ml) (priming) and then incubated (activation) with Nigericin (5 μM), MSU crystals (MSU; 250 μg/ml), Alum Hydroxide (150 μg/ml), transfected Poly (dA:dT) (1 μg/ml), or E. coli outer membrane vesicles (OMVs) (100 μg/ml). After 24h, the secretion of mature human (h)IL-1β was assessed in the culture supernatant using HEK-Blue™ IL-1β sensor cells, which express an NF-κB SEAP reporter gene. QUANTI‑Blue™ Solution was used to measure SEAP activity. Optical density (OD) was read at 630 nm.

~3x10⁵ THP1-Null2 (blue) and THP1‑KO-ASC cells (red) were incubated for 3h at 37°C with LPS-EK (1 μg/ml) (priming) and then incubated (activation) with Nigericin (0.15-10 μM). After 6h, cell death was assessed using the lactate dehydrogenase (LDH) assay.

Quantitative RT-PCR analysis showing the fold change of NLRP3 and ASC genes in THP1-defASC cells compared to THP1-null cells.

Cells were primed with LPS (1 μg/ml) and then induced (activated) with ATP (5 mM) or MSU crystals (MSU; 100 μg/ml). After 24h incubation,  the secretion of mature IL-1β was assessed in the culture supernatants using HEK-Blue™ IL-1β sensor cells expressing an NF-κB SEAP reporter gene. QUANTI‑Blue™ Solution was used to measure SEAP activity. Optical density (OD) was read at 630 nm.

Specifications

THP1-KO-ASC cells

Antibiotic resistance: Zeocin®

Growth medium: RPMI 1640, 2 mM L-glutamine, 25 mM HEPES, 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, 100 µg/ml Normocin™

Quality Control:

• Biallelic ASC knockout has been verified by PCR, DNA sequencing, Western blot (WES™), and functional assays.
• The stability for 20 passages, following thawing, has been verified. 
• These cells are guaranteed mycoplasma-free. 

THP1-defASC cells

Antibiotic resistance: Hygromycin B

Growth Medium: RPMI 1640, 2 mM L-glutamine, 25 mM HEPES, 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, 100 μg/ml Normocin™

Quality control:

• ASC deficiency (def) has been verified by qRT-PCR and functional assays.
• The stability for 20 passages, following thawing, has been verified. 
• These cells are guaranteed mycoplasma-free. 

Both of these products are covered by a Limited Use License (See Terms and Conditions).

Contents

THP1-KO-ASC cells

• 3-7 x 10⁶ THP1-KO-ASC cells in a cryovial or shipping flask
• 1 ml of Zeocin® (100 mg/ml). Store at 4 °C or at -20 °C.
• 1 ml of Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria, and fungi.

Shipped on dry ice (Europe, USA & Canada)

THP1-defASC cells

• 3-7 x 10⁶ THP1-defASC cells in a cryovial or shipping flask
• 1 ml of Hygromycin B Gold (100 mg/ml)
• 1 ml of Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria, and fungi.

Shipped on dry ice (Europe, USA & Canada)

Details

Inflammasomes are multimeric protein complexes that are crucial for host defense against infection and response to endogenous danger signals. The canonical inflammasome response is driven by aggregation of a sensor (i.e. NLRP3) with the ASC adaptor and pro-caspase-1. Activation of caspase-1 (CASP1) induces the maturation of pro-IL-1β/pro-IL-18 and cleavage of the pore-forming protein gasdermin D (GSDMD), leading to secretion of IL-1β/ 18 and pyroptosis. 

ASC is essential to canonical inflammasome sensors that do not contain a CARD domain, such as NLRP3, AIM2, and Pyrin [1]. ASC's bipartite composition, consisting of one PYD and one CARD domain, allows the recruitment of the CARD-containing pro-caspase-1 to these sensors.  The NLRP1 and NLRC4 inflammasome sensors have a CARD domain, and can thus recruit pro-caspase-1 either directly, or through ASC. However, NLRP1 or NLRC4 activation in the absence of ASC triggers a reduced secretion of mature IL-1β and IL-18 [1]. Importantly, non-canonical inflammasomes (i.e. CASP4/5/11) activate CASP1 indirectly: their activation triggers GSDMD-driven release of alarmins and K+ efflux, which in turn, induce NLRP3- and CASP1-mediated IL-1β/-18 maturation and secretion. Therefore, the absence of ASC affects the downstream inflammatory signaling from the non-canonical inflammasome also. 

In resting cells, ASC is present in a soluble and diffuse form both in the cytoplasm and nucleus [2]. Inflammasome activation in most cells leads to the formation of one large, micrometer-sized, ASC ‘speck’ per cell, thus concentrating CASP1 activation sites [2,3]. Yet, the number of ASC specks may dependent on the inflammasome inducer used. For example, Nigericine promotes the formation of numerous specks, whereas ATP leads to the accumulation of fewer specks [4].

1. Mathur A. et al., 2017. Molecular mechanisms of inflammasome signaling. J. Leuk. Biol. 103:233.
2. Hoss F. et al., 2017. Assembly and regulation of ASC specks. Cell. Mol. Life Sci. 74:1211. 
3. Stutz A. et al., 2013. ASC speck formation as a readout for inflammasome activation. Methods Mol. Biol.
4. Zha Q. et al., 2016. ATP-induced inflammasome activation and pyroptosis is regulated by AMP-activated protein kinase in macrophages. Front Immunol. 7:597.

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