TNFalpha- eGFP -dsRED Canonical Pathway Differentiation Measured in Cell-Based System Using Flow Cytometry - 2122-08_Inhibitor_Dose_DryPowder_Activity
Keywords: NF-kappaB, Epstein-Barr Virus, inhibitor, LMP1, Latent Membrane Protein 1, luciferase reporter, TES1, TES2, TNF-alpha FACS ..more
BioActive Compounds: 14
Keywords: NF-kappaB, Epstein-Barr Virus, inhibitor, LMP1, Latent Membrane Protein 1, luciferase reporter, TES1, TES2, TNF-alpha FACS
Assay Overview: Epstein-Barr Virus is a ubiquitous Herpesvirus that is an important cause of Hodgkin's Disease, other Lymphoproliferative Diseases, and Nasopharyngeal Carcinoma. EBV infection mimics NF-kB hyperactivation states present in many malignancies. The EBV oncoprotein LMP1 constitutively activates both canonical and noncanonical NF-kB pathways in a ligand-independent fashion. LMP1 is expressed in most EBV-associated lymphoproliferative and epithelial malignancies. LMP1 activates NF-kB via two cytoplasmic signaling domains. The membrane proximal "TES1" domain activates a non-canonical NF-kappaB pathway, while the membrane distal "TES2" domain activates canonical NF-kappaB.
This secondary assay uses the same transfected HEK293 cell line as the primary screen, with a doxycycline & 4-hydroxytamoxifen inducible LMP1 TES2 construct and a NF-kappaB luciferase reporter as well as an eGFP reporter. In addition, a consitutive dsRed protein is expressed to assess cell viability and expression levels. However, in this case induction of the system occurs using TNFa rather than through expression of the TES2 construct. This will activate the canonical NF-kappaB pathway and result in expression of the eGFP reporter gene. Inhibitors of the canonical pathway will block expression of the reporter construct. In this case the readout uses a Fluorescent Activated Cell Sorter to count the number of cells expressing TNF-induced green fluorescence; In this dataset only the GFP curves are reported, singal windows vs. dsRed were calculated separately.
Expected Outcome: Inhibitors of the NF-kB canonical pathway will show a reduction in green fluorescence but no reduction in the constitutive red fluorescence; A window of at least 2-fold is desired.. To be of interested as a probe, compounds should be specific to either the canonical or non-canonical pathway and should therefore show no activity in either this assay or in an IL-1beta induced non-canonical pathway reporter. Compounds that inhibit both are likely inhibiting at the NF-kB level (such as by inhibiting degradation as occurs with MG-132 or IKK-inhibitor VIII) and are therefore unlikely to be specific to one of the pathways related to EBV induced hyperactivation.
LMP1 secondary assay: TNFa stimulated eGFP reporter/dsRed control with FACS readout
Day 0, cell grown in T175 TC flask to 95% confluence to yield 30 Million (TrypLE phenol free) and resuspended to dispensing at 125,000 cells / mL of phenol free DMEM
Day 1, plate cells 5000 per well in 40 uL media (phenol red free DMEM/10% Tet Free FBS/Pen/Strep/L-Glutamine); incubate in standard TC conditions (5% CO2; 95% humidity, 37C) for 24 hours.
Day 2, add 10 uL per well of stimulant (1ng/mL TNFa) with a Combi multidrop (Thermo);
add 100 nL 3.75 mM dry powder cherry picked into 40 uL assay volume in white, opaque Corning 8867 barcoded 384 well plates using a pin tool (CyBio). MG132 (Calbiochem 474790), a proteasome inhibitor that blocks degradation of NF-kappaB inhibitor I-kappa-B-alpha, was added to positive control wells to a final concentration of 16.7 uM. Additionally, IKK-2 Inhibitor VIII (16.7 uM, Calbiochem #401487) was included as another control specific for the NF-kB pathway, and Bay 11-7082 (16.7 uM, Sigma #B5556) was included as a generally toxic control.
Incubate 16 hours at 37 degrees C in Liconic incubator, 95% humidity 5% CO2.
Day 3, Plates were gently washed twice with PBS, then trypsinized with 10 uL of trypsin for 5 minutes at 37, then 10 uL of DMEM/10% FCS was added to quench trypsin. Single cell suspensions were produced by manual pipetting (10 times per well), using a multi-channel pipetteman. Approximately 3000 cells per well were counted on a Becton Dickinson (BD) LSRII FACS in 384 well sampling mode. FITC excitation voltage was 350 and PE voltage was 375. FITC filters were 505LP mirror -> 530/30nm emission (515-545 emission). Red channel (Phycoerythrin) filters were 550 LP mirror, 575/26nm emission (562-588 nm emission). To avoid bias, only live cells were counted, as defined by the forward/side scatter gates with forward (FSC) and side scatter (SSC) settings of 290 and 265, respectively. The median GFP and dsRED values from each well were used for analysis.
PRESENCE OF CONTROLS: Neutral control wells (NC; n=38) and positive control wells (PC; n=10) were included on every plate.
EXPECTED OUTCOME: Active compounds result in decreasing readout signal.
ACTIVE CONCENTRATION LIMIT:
For each sample, the highest valid tested concentration (Max_Concentration) was determined and the active concentration limit (AC_limit) was set to equal (2)(Max_Concentration).
The raw signals of the plate wells were normalized using the 'Neutral Controls' method in Genedata Assay Analyzer (v10.0.2):
The median raw signal of the intraplate neutral controls (NC) is set to a normalized activity value of 0.
A normalized activity value of 100 is defined as (2)(NC).
A normalized activity value of -50 is defined as (0.5)(NC).
Experimental wells values were scaled to this range.
PATTERN CORRECTION: No plate pattern correction algorithm from Genedata Condoseo (v.10.0.2) was applied.
MEASUREMENT USED TO DETERMINE ACTIVE CONCENTRATION (AC): absACnn, the concentration at which the curve crosses threshold -35.0
AC values were calculated using the curve fitting strategies in Genedata Screener Condoseo (7.0.3).
AC values were calculated up to the active concentration limit described for each sample.
pAC was set to equal -1*log10(AC)
Activity_Outcome = 1 (inactive) when:
a) compound shows activity but in a direction opposite to the expected outcome
in these cases, values describing curve fitting parameters (Sinf, S0, Hill Slope, log_AC50, log_AC50_SE) are set to null
b) curve fit is constant where activity is > -30% and < 30% at all tested concentrations, or
c) AC > AC_limit
Activity_Outcome = 2 (active) when:
AC <= AC_limit
Activity_Outcome = 3 (inconclusive) when:
a) Curve fitting strategy resulted in a constant fit with activity >= -70% but <= -30%, or
b) The fit was deemed not valid due to poor fit quality.
If PUBCHEM_ACTIVITY_OUTCOME = 1 (inactive) or 3 (inconclusive),
then PUBCHEM_ACTIVITY_SCORE = 0
If PUBCHEM_ACTIVITY_OUTCOME = 2 (active)
then PUBCHEM_ACTIVITY_SCORE = (10)(pAC)
Scores relate to AC in this manner:
120 = 1 pM
90 = 1 nM
60 = 1 uM
30 = 1 mM
0 = 1 M
When the active concentration (AC) is calculated to be greater than the highest valid tested concentration (Max_Concentration), the PUBCHEM_ACTIVITY_SCORE is calculated using Max_Concentration as the basis.
When the active concentration (AC) is calculated to be less than the lowest tested concentration, the PUBCHEM_ACTIVITY_SCORE is calculated using the lowest tested concentration as the basis.
The individual dose data point columns ('Activity_at_xxuM') reported here represent the median of valid (unmasked) replicate observations at each concentration. These values are the inputs to a curve fitting algorithm.
All other data columns represent values which are derived during the curve fitting algorithm; this may sometimes include automatic further masking of some replicate data points.
Occasionally this results in perceived inconsistencies: for example, between the derived 'Maximal_Activity' and the apparent most active data point.
Categorized Comment - additional comments and annotations
* Activity Concentration. ** Test Concentration.
Data Table (Concise)