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

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

Assay: Cell viability of BJ-TERT (BJ fibroblasts transformed with hTERT.) Using CellTiterGlo, which measures the amount of intracellular ATP from living 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, which do not contain oncogenic HRAS, are working through a mechanism that is not of interest for the goals this project.

Positive control: RSL3, from Stockwell lab, 32 mg, assigned BRD-K56411643-001-01-8; Erastin, Batch1: Sigma E7718 assigned BRD-A25004090-001-01-9 or Batch2: Calbiochem 329600, assigned BRD-A25004090-001-02-7

Cell Passage
1. BJ-TERT cells (Stockwell lab) are normally maintained in 35mL of media in a T175 cell culture flask, which is kept in a TC incubator at 95% humidity, 5% CO2, 37 degrees C.
2. To passage, harvest the cells by first aspirating the media and rinsing the flask with 10 mL PBS. Aspirate the PBS and add 5 mL trypsin to the flask. Incubate with the trypsin for 5 minutes in the TC hood (22 degrees C).
3. Add 8 mL of media to the trypsin. Dislodge the cells from the bottom of the flask by pipeting. Remove an aliquot of cells for counting. Cell density should be between 0.5 and 1 x 10^6/mL.
4. Transfer 4.5 x 10^6 (~2 mL) of the 13 mL cells in trypsin/media to 33 mL fresh media in a T175 for harvest at ~90% confluence two days later, or 1.5 x 10^6 (~1 mL)34 mL fresh media for harvest three days later.
5. Lines are stopped at P20 and restarted from an earlier frozen aliquot.

Assay Protocol
Day -1
1. Harvest the cells by first aspirating the media and rinsing the flask with 10 mL PBS. Aspirate the PBS and add 5 mL trypsin to the flask. Incubate with the trypsin for 5 minutes in the TC hood (22 degrees C).
2. Add 8 mL of media to the trypsin. Dislodge the cells from the bottom of the flask by pipeting. Remove an aliquot of cells for counting.
3. Add media to adjust the concentration of the cells to 33,000/mL and add a sterile stir bar to the flask.
4. Plate the cells to 384-well plates using the Combi (walkup room), transferring 30 uL to each well, for a total of 1,000 cells/well. Keep the cell flask on a stir plate, gently spinning, to maintain a uniform cell suspension during the plating operation.
5. Incubate the plates overnight in a TC incubator at 95% humidity, 5% CO2, 37 degrees C.
Day 1
6. Transfer the plates to an online Liconic incubator at 95% humidity, 5% CO2, 37 degrees C.
7. Pin with 50 nL compound (BL2+ system, one dip) using 25nL head programmed to deliver 50 nL, or 100 nL using 100nL head as appropriate.
8. Return plates to the Liconic incubator at 95% humidity, 5% CO2, 37 degrees C for overnight incubation.
Day 2
9. Transfer the plates to offline TC incubators, 95% humidity, 5% CO2, 37 degrees C. (This step is done to free up working space in the Liconic incubator.)
Day 3
10. Move plates to racks in a Liconic incubator (on GS system), 95% humidity, 5% CO2, 37 degrees C. Load plate racks so that all plates will be pulled for the read run in the same order that they were pinned.
11. Initiate the automation protocol to start the read run. Plates are moved from the Liconic incubator to the GS carousel at normal room conditions (ambient humidity, 22 degrees C). Plates are allowed to cool on the carousel for 30 minutes, lids on.
12. Each plate is delidded and 30 uL Cell Titer Glo (prepared as 1:3 dilution) is added to each well using the Combi.
13. Plates are incubated an additional 10 minutes in the carousel without lids.
14. Plates are read with the Envision, luminescence detection, read time = 0.1 second.
15. Plates are relidded and discarded.

Dose Response Data Analysis:
32 Negative control (DMSO) 16 positive control 1 (Erastin), 16 positive control 2 (RSL3) were included on each plate.

All wells tested on a plate were background subtracted using the median of the negative controls well values on the same plate. All wells tested on a plate were scaled relative to the median value of the same-plate positive control wells, using only the control more active for the tested cell line. Plate pattern correction was not used.

IC50 values were calculated using the Smart Fit strategy of Genedata
Screener Condoseo (v6.0.1). EC50 values were extrapolated up to 1 log
over the highest tested concentration.

PUBCHEM_ACTIVITY_SCORE
Inactive compounds = 0
Active compounds = -10*Log(EC50)

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

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

Activity_Outcome = 3 (inconclusive)
Constant fit curve actvitity is >30% but <70%
or
Constant fit curve is labelled as invalid due to large fit error

Grant Number: MH084117-01