Human STING (R232)-dependent STAT6 reporter cells

STING-dependent STAT6 activation
in HEK-Blue™ STAT6-hSTING-R232 cells

Human STING-dependent STAT6 reporter HEK293 cells

HEK-Blue™ STAT6-hSTING-R232 cells are specifically designed to monitor the induction of STAT6-dependent signaling upon activation of STING. These cells were generated by stable overexpression of the most prevalent human (h)STING variant R232 [1], in a human embryonic kidney (HEK)293-derived cell line that expresses human STAT6 and a STAT6-inducible secreted embryonic alkaline phosphatase (SEAP) reporter. STAT6-dependent SEAP activity is readily assessable in the supernatant using QUANTI-Blue™ Solution, a detection reagent.

STING Background

STING (stimulator of interferon genes) is essential in the effective immune response against foreign or self-DNA through the sensing of cytoplasmic cyclic dinucleotides (CDNs) [2]. The activation of STING causes a TANK-binding-kinase-I (TBK1)-dependent cascade, ultimately, leading to IFN regulatory factor (IRF3)-dependent type I IFN production and NF-κB-dependent inflammatory cytokine production [2]. Additionally, signal transducer and activator of transcription 6 (STAT6) has been reported to be recruited to STING for TBK1-mediated phosphorylation during viral infection [3]. Ultimately, the STING‑dependent activation of STAT6 induces a specific set of anti-viral chemokines responsible for immune cell homing, which leads to reduced viral replication [3]. Notably, this specific 'viral' activation of STAT6 was found to be Janus kinase (JAK)-independent and is thus, distinct from the ‘canonical’ STAT6 pathway activated by IL-4 and IL-13, which is critical in adaptive immunity [3].

Features of HEK-Blue™ STAT6-hSTING-R232 cells:

• Verified overexpression of hSTING (PCR and functional assays)
• Functionally validated with a selection of STING ligands
• These cells do not respond to Type I IFNs
• Readily assessable STAT6-dependent SEAP reporter activity
• The stability for 20 passages has been verified
• Guaranteed mycoplasma-free

Applications for HEK-Blue™ STAT6-hSTING-R232 cells:

• Studying the role of STING-dependent STAT6 activation in response to viral infection

References:

1. Yi, G. et al. 2013. Single nucleotide polymorphisms of human STING can affect innate immune response to cyclic dinucleotides. PLoS One 8, e77846.
2. Cheng, Z. et al. 2020. The interactions between cGAS-STING pathway and pathogens. Signal Transduct Target Ther 5, 91.
3. Chen, H. et al. 2011. Activation of STAT6 by STING is critical for antiviral innate immunity. Cell 147, 436-446.

Figures

STAT6 activation upon STING induction. HEK-Blue™ STAT6-hSTING-R232 and their parental cells (STAT6 reporter cells) were incubated with 2’3’-cGAMP (30 μg/ml), 2’3’-cGAM(PS)2 (Rp/SP) (ADU-S100; 30 μg/ml), cAIM(PS)2 Difluor (Rp/Sp) (30 μg/ml), 3’3’-cGAMP (30 μg/ml), or DMXAA (30 μg/ml) in cell culture medium. After overnight incubation, the STAT6 response was assessed by measuring the activity of SEAP in the supernatant using QUANTI-Blue™ Solution, a SEAP detection reagent. Data are presented fold change over non-induced cells (mean ± SEM).

Specifications

Antibiotic resistance: Blasticidin, Hygromycin B, and Zeocin®

Growth medium: DMEM, 4.5 g/l glucose, 2 mM L-glutamine,10% (v/v) heat-inactivated fetal bovine serum (FBS), Pen‑Strep (100 U/ml-100 μg/ml), 100 μg/ml Normocin™

Quality Control:

• STAT6-dependent SEAP reporter activity in response to various STING ligands and other cytokines has been validated.
• The stability for 20 passages, following thawing, has been verified. 
• These cells are guaranteed mycoplasma-free. 

Contents

• 3-7 x 10⁶ HEK-Blue™ STAT6-hSTING-R232 cells in a cryovial or shipping flask
• 2 x 1 ml of HEK-Blue™ Selection (250x concentrate)
• 1 ml of Normocin™ (50 mg/ml). Normocin™ is a formulation of three antibiotics active against mycoplasmas, bacteria, and fungi.
• 1 ml of QB reagent and 1 ml of QB buffer (sufficient to prepare 100 ml of QUANTI-Blue™ Solution, a SEAP detection reagent)

 Shipped on dry ice (Europe, USA, Canada and some areas in Asia)

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