AID	Name	Type	Comment
1554	MLPCN Ras selective lethality-BJeLR viability	screening	Primary HTS
1674	Broad Institute MLPCN Ras Selective Lethality Project	summary	Summary

Primary Collaborators(and laboratory where this assay was performed):
Brent Stockwell,Columbia University,bs2198@columbia.edu,212-854-2948,614 Fairchild Center, MC 2406, 1212 Amsterdam Ave, New York, NY 10027
Dan Zaharevitz,NCI Science Officer,zaharevd@mail.nih.gov

Keywords: Ras, apoptosis, cancer, VDAC, oxidative cell death

Assay: Cell viability of BJeLR (BJ fibroblasts transformed with hTERT, genomic SV40 LT and ST oncoproteins, and oncogenic HRASV12). Using Alamer Blue, which is metabolized to a fluorescent product by live cells, the total amount of viable cells can be measured by luminescence readout. Cells are treated with compounds for 48 hours to allow detection of compounds that may be slower-acting.

Expected Outcome: Compounds that are toxic to these transformed fibroblasts, either selectively due to the HRAS oncogene or due to nonspecific toxicity, will cause a loss of fluorescence signal due to fewer live cells.

Assay Protocol:

Growth medium:
730 ml DMEM with 4mM L-glutamine (Gibco # 11995)
210 ml M199 (Sigma #M7528)
150 ml heat inactivated Fetal Bovine Serum (Gibco # 26140-079)
10 ml Penicillin/Streptomycin (Gibco #15140-122)

1. Make Daughter plates
a. Transfer 148uL of BJ media to empty Daughter plates
b. Transfer 2ul of cmpd solution in Mother plates to media-filled Daughter plates (1:75 dilution) and mix thoroughly
2. Make Step-Daughter plates
*Both Daughter and Step-Daughter plates are the same Greiner #781270, 384 cone deep

a. Fill empty Step-Daughter plate with 75uL BJ media except col3 and col13
b. Transfer 150uL of hits from Daughter plate to col3 and col13
- We fill C3 through N3 and C13 through N13; the total number of primary hits in a single Step-Daughter plate is 24 (12 on col3 and 12 on col13)
c. Make 2-fold dilution series by transferring 75uL to the next column (from col3 to col12 and col13 to col22)

3. Seed cells & add compound to Assay plates
a. Prepare BJeH cell solution
b. Transfer 36uL of BJeH cell solution to empty Assay plates; the concentration of BJeH cells in the Assay plate is 1000 cells/well
c. Transfer 4ul of cmpd solution from Step-Daughter plate to cell-seeded Assay plates (1:10 dilution final concentration of cmpd is from 0.01ug/mL to 5.33 ug/ml in 2-fold dilution series)

4. Incubate cells for 48h at 37C/5%CO2

5. Add alamar blue solution to the Assay plates
a. Transfer 10ul of 50% alamar blue in BJ growth media to the Assay plates
b. Incubate ~16 hours before reading plates

6. Read Fluorescence at 544 nM excitation, 590 nM emission

Powder Dose Data Analysis

Normalized data for each compound was manually imported into Genedata Condoseo (v 7.0.3) from external sources, and dose response curves were fit using the available algorithms (Smart Fit, etc.).


The PUBCHEM_ACTIVITY_SCORE was set to:
inactive compounds -> 0
active compounds -> -10*log(EC50)

The PUBCHEM_ACTIVITY_OUTCOME was set to:

Activity_Outcome = 1 (inactive) when
EC50 > 1 log over the highest tested concentration

Activity_Outcome = 2 (active) when
EC50 <= 1 log over the highest tested concentration

Activity_Outcome = 3 (inconclusive) when
Curve fitting strategy resulted in a constant fit with activity >30% but <70% ('undefined' in Genedata)
or
The fit was not valid due to poor fit quality.

Grant Number: MH084117-01