NF-κB-dependent Luciferase Activation and Quantification of Gene Expression in Salmonella Infected Tissue Culture Cells

Summary

Here, we present a protocol to quickly and easily measure nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation in cell lines expressing NF-κB::luciferase reporter constructs, via measurements of luminescence in the cell lysate. Additionally, gene expression is determined via RT-qPCR isolated from cells infected with Salmonella Typhimurium.

Abstract

The dimeric transcription factor NF-κB regulates many cellular response pathways, including inflammatory pathways by inducing the expression of various cytokines and chemokines. NF-κB is constitutively expressed and is sequestered in the cytosol by the inhibitory protein nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha (IκBα). Activation of NF-κB requires the degradation of IκBα, which then exposes a nuclear localization signal on NF-κB and promotes its trafficking to the nucleus. Once in the nucleus, NF-κB binds to the promotor region of NF-κB target genes such as interleukin 6 (IL-6) and IL-23, to promote their expression.

The activation of NF-κB occurs independently of transcription or translation. Therefore, the activation state of NF-κB must be measured either by quantifying NF-κB specifically in the nucleus, or by quantifying expression of NF-κB target genes. In this protocol, cells stably transfected with an NF-κB::luciferase reporter construct are assayed for NF-κB activation using in vitro tissue culture techniques. These cells are infected with Salmonella Typhimurium to activate NF-κB, which traffics to the nucleus and binds to κB sites in the promoter region of luciferase, inducing its expression. Cells are lysed and analyzed with the luciferase assay system. The amount of luciferase produced by the cells correlates with the intensity of the luminescence signal, which is detected by a plate reader. The luminescence signal generated by this procedure provides a quick and highly sensitive method by which to assess NF-κB activation under a range of conditions. This protocol also utilizes quantitative reverse transcription PCR (RT-qPCR) to detect relative mRNA levels that are indicative of gene expression.

Introduction

The nuclear factor-κB (NF-κB) family of proteins are important transcription activators that regulate gene expression in various biological pathways. Activation of NF-κB induces transcription of target genes, many of which are important for immune and inflammatory responses, cell proliferation, stress responses and cancer progression^1,2. NF-κB plays an integral role in mediating early inflammatory outcomes for pathogen clearance. Given the many biological processes mediated by NF-κB activation, disruptions in its signaling can have serious consequences for health and disease. Loss of f....

Protocol

1. Cell Passaging and Seeding

1. Maintain HeLa 57A cells in a 75 cm² flask containing 10 mL of Dulbecco's modified Eagle's media (DMEM) supplemented with 5% heat inactivated fetal bovine serum (FBS) at 37 °C in a 5% CO₂ incubator.
2. Aspirate cell culture media and wash with 1 mL of 0.05% trypsin-EDTA solution. Aspirate trypsin and replace with an additional 1 mL. Place the flask in a 37 °C incubator for 4-5 min to allow cells to detach from flask.
3. Add 9 mL of cell culture media and gently wash the bottom of the flask a few times to dislodge cells and form a homogenous suspension.
4. Dilute HeLa ....

Results

The assay described here focuses on activation of the transcription factor NF-κB using a NF-κB-dependent luciferase reporter that is stably transfected into a line of HeLa cells. Activated NF-κB translocates to the nucleus where it binds κB binding sites of target genes, including the pro-inflammatory cytokines IL6 and IL23. A general overview of NF-κB activation is depicted in Figure 1. The gram-negative bacterium Salmonella enterica

Discussion

The major contribution of the protocol described is that it provides a fast and easy method to detect NF-κB activation in cells, which allows for high throughput analysis of multiple stimulatory conditions or drugs affecting NF-κB activation. Here, we describe a protocol for NF-κB activation in Salmonella-infected HeLa cells. These cells can be used for infection with other pathogens as well to study the impact of bacterial infection on NF-κB activation. In addition, NF-κB-dependent luci.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Adhesive film	VWR International	60941-070
Chloroform	Fisher Bioreagents	C298-500
DMEM	Thermo Fisher	11665092
DNAse treatment kit	Qiagen	79254
dNTPs	Promega	U1511
Ethanol	Fisher Bioreagents	BP2818100	molecular grade
FBS	Sigma-Aldrich	F0926
HeLa 57A cells			Ref # 15
High-Capacity cDNA Reverse Transcription Kit	Applied Biosystems	4368814
Isopropanol	Fisher Bioreagents	BP26181
Kanamycin	Fisher Bioreagents	BP906-5
LB agar	Fisher Bioreagents	BP1425-500
Lysogeny broth	Fisher Bioreagents	BP1426-500
MgCl2	Fisher Chemical
NanoDrop ND-1000	Thermo Scientific		spectrophotometer
Promega luciferase assay system	Promega	E1501	Cell lysis buffer & luciferin substrate
Random Hexamers	Thermo Scientific	SO142
Real-time GAPDH forward primer			5'-CCAGGAAATGAGCTTGAC AAAGT-3'
Real-time GAPDH reverse primer			5-'CCCACTCCTCCACCT TTGAC-3'
Real-time IL-23 forward primer			5-'GAGCCTTCTCTGCTCCC TGAT-3'
Real-time IL-23 reverse primer			5'-AGTTGGCTGAGGCCCAGTAG-3'
Real-time IL-6 forward primer			5'-GTAGCCGCCCCACACAGA-3'
Real-time IL-6 reverse primer			5'-CATGTCTCCTTTCTCAGG GCTG-3'
Reverse Transcriptase	Applied Biosystems	4308228
RNAse inhibitor	Thermo Scientific	EO0381
RT buffer	Promega	A3561
SL1344			Ref # 17
SL1344 ΔsipA sopB::MudJ sopE2::pSB1039			Ref # 18
SL1344 ΔsopE ΔsipA sopB::MudJ sopE2::pSB1039			Ref # 18
SYBR green	Applied Biosystems	4309155	2x mastermix
Tri-reagent	Molecular Research Center	TR 118	guanidine thiocyanate
Trypsin -EDTA	Thermo Fisher	25300054	0.05% Trypsin-EDTA
Ultrapure water	Fisher Bioreagents	BP248450
Well plate for PCR	VWR International	89218-294	384-well plate