A Genome-wide Haploid Genetic Screen Identifies Regulators of Glutathione Abundance and Ferroptosis Sensitivity

. Author manuscript; available in PMC: 2019 Mar 19.

Published in final edited form as: Cell Rep. 2019 Feb 5;26(6):1544–1556.e8. doi: 10.1016/j.celrep.2019.01.043

KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Rat monoclonal anti-MRP1	Abcam	Cat# ab3368; RRID: AB_303746
Rabbit polyclonal anti-NRF2	Abcam	Cat# ab137550; RRID: AB_2687540
Mouse monoclonal anti-KEAP1	Sigma-Aldrich	Cat# K2769; RRID: AB_10602870
Rabbit polyclonal anti-GCLM	Novus Biologicals	Cat# NBP1-33405; RRID: AB_2107841
Mouse monoclonal anti-Tubulin alpha, clone DM1A	Fisher Scientific	Cat# MS581P1; RRID: AB_144075
IRDye 680LT Goat anti-Rat IgG	LI-COR	Cat# 925-68076
IRDye 800CW Goat anti-Rat IgG	LI-COR	Cat# 925-32219
IRDye 800CW Donkey anti-Rabbit IgG	LI-COR	Cat# 926-32213
IRDye 800CW Donkey anti-Mouse IgG	LI-COR	Cat# 926-32212
IRDye 680LT Donkey anti-Rabbit IgG	LI-COR	Cat# 926-68023
IRDye 680LT Donkey anti-Mouse IgG	LI-COR	Cat# 926-68022
Bacterial and Virus Strains
NucLight Red lentivirus reagent (EF1a, Puromycin) Nuclear-localized mKate2 (Nuc::mKate2)	Essen BioSciences	Cat# 4265
NucLight Red lentivirus reagent (EF1a, Bleomycin) Nuclear-localized mKate2 (Nuc::mKate2)	Essen BioSciences	Cat# 4627
Chemicals, Peptides, and Recombinant Proteins
SYTOX Green	Life Technologies	Cat# S7020
DFO mesylate	Cayman Chemical	Cat# 14595; CAS: 138-14-7
Cyst(e)inase	Cramer et al., 2017	N/A
Erastin2 (Compound 35MEW28 in Dixon et al., 2014)	Dixon et al., 2014	N/A
Buthionine sulfoximine	Fisher Scientific	Cat# AC23552-0010; CAS: 83730-53-4
Q-VD-OPh	Fisher Scientific	Cat# OPH00101M
Puromycin	Life Technologies	Cat# A11138-03
SYTOX Green	Life Technologies	Cat# S7020
Lipofectamine LTX Reagent with PLUS Reagent	Life Technologies	Cat# 15338-100
C11 BODIPY 581/591 (4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid	Molecular Probes	Cat# D3861
Concanavalin A-Alexa Fluor 350	Molecular Probes	Cat# C11254
261 member bioactive chemical library	Selleck Chemicals	Cat# L2000
BAY-11-7821/7082	Selleck Chemicals	Cat# S2913; CAS: 19542-67-7
BAY-11-7085	Selleck Chemicals	Cat# S7352; CAS: 196309-76-9
RSL3	Selleck Chemicals	Cat# S8155; CAS: 1219810-16-8
Doxorubicin hydrochloride	Selleck Chemicals	Cat# S1208; CAS: 25316-40-9
Vincristine sulfate	Selleck Chemicals	Cat# S1241; CAS: 2068-78-2
Bortezomib	Sigma-Aldrich	Cat# NC0587961; CAS: 179324-69-7
N-Acetyl-L-Cysteine	Sigma-Aldrich	Cat# A8199; CAS: 616-91-1
Thapsigargin	Sigma-Aldrich	Cat# T9033; CAS: 67526-95-8
Ferrostatin-1	Sigma-Aldrich	Cat# SML0583; CAS: 347174-05-4
Polybrene	Sigma-Aldrich	Cat# H9268; CAS: 28728-55-4
Acivicin	Sigma-Aldrich	Cat# SML0312; CAS: 42228-92-2
¹³C₅-L-glutamine	Sigma-Aldrich	Cat# 605166; CAS: 184161-19-1
PolyJet In Vitro DNA Transfection Reagent	SigmaGen Laboratories	Cat# SL100688
Monochlorobimane	Thermo Fisher	Cat# M-1381MP; CAS: 76421-73-3
Critical Commercial Assays
QuickExtract DNA Extraction Solution	Epicenter	Cat# QE09050
QIAshredder RNA Extraction Column Kit	QIAGEN	Cat# 79654
RNeasy Plus RNA Extraction Kit	QIAGEN	Cat# 74134
TaqMan Reverse Transcriptase Kit	TaqMan	Cat# N8080234
Cayman Glutathione Kit	Cayman Chemical	Cat# 703002
QIAprep Spin Miniprep Kit	QIAGEN	Cat# 27106
QIAquick PCR Purification Kit	QIAGEN	Cat# 28106
SYBR Green Master Mix	Life Technologies	Cat# 4367659
Gateway LR Clonase II Enzyme Mix	Thermo Fisher	Cat# 11791-020
BCA Protein Assay Kit	Thermo Fisher	Cat# 23225
PrestoBlue Cell Viability Reagent	Thermo Fisher	Cat# A13261
Bradford Assay Kit	Bio-Rad	Cat# 5000002
Experimental Models: Cell Lines
Human: HAP1	Carette et al., 2011b	N/A
Human: HAP1^N	This paper	N/A
Human: HAP1 Control^A	Horizon Discovery	Cat# C631
Human: HAP1 KEAP1^KO	Horizon Discovery	Cat# HZGHC003774c005
Human: HAP1 Control^B	This paper	N/A
Human: HAP1 MRP1^KO1	This paper	N/A
Human: HAP1 MRP1^KO2	This paper	N/A
Human: HAP1 MRP1^KO1 + CMV-empty	This paper	N/A
Human: HAP1 MRP1^KO1 + CMV-MRP1	This paper	N/A
Human: HAP1 MRP1^KO1 + CMV-MRP11^K332L	This paper	N/A
Human: HAP1 NAA38^KO1	This paper	N/A
Human: HAP1 NAA38^KO2	This paper	N/A
Human: HAP1 GSTO^KO1	This paper	N/A
Human: HAP1 GSTO^KO2	This paper	N/A
Human: HAP1 SETD5^KO1	This paper	N/A
Human: HAP1 SETD5^KO2	This paper	N/A
Human: A549	ATCC	Cat# CCL-185; RRID: CVCL_0023
Human: A549^N	Forcina et al., 2017	N/A
Human: A549^N MRP1^KO1	This paper	N/A
Human: A549^N MRP1^KO2	This paper	N/A
Human: HT-1080	ATCC	Cat# CCL-121; RRID: CVCL_0317
Human: HT-1080^N	Forcina et al., 2017	N/A
Human: U-2 OS	ATCC	Cat# HTB-96; RRID: CVCL_0042
Human: U-2 OS^N	Forcina et al., 2017	N/A
Human: T98G^N	Forcina et al., 2017	N/A
Human: NCI-H1299 (called H1299)	ATCC	Cat# CRL-5803; RRID: CVCL_0060
Human: H1299^N	This paper	N/A
Human: U-2 OS + CMV-empty	This paper	N/A
Human: U-2 OS + CMV-MRP1^K332L	This paper	N/A
Human: U-2 OS + CMV-MRP1^K332L	This paper	N/A
Human: U-2 OS^N + CMV-empty	This paper	N/A
Human: U-2 OS^N + CMV-MRP1	This paper	N/A
Human: H1299 + CMV-empty	This paper	N/A
Human: H1299 + CMV-MRP1	This paper	N/A
Human: H1299 + CMV-MRP1^K332L	This paper	N/A
Human: H1299^N + CMV-empty	This paper	N/A
Human: H1299^N + CMV-MRP1	This paper	N/A
Oligonucleotides
See Table S2.		N/A
Recombinant DNA
pSpCas9 (BB)-2A-GFP-sgRNA-p53	This paper	N/A
pLenti-CMV-Puro-MRP1	This paper	N/A
pLenti-CMV-Puro-MRP1K332L	This paper	N/A
Other Plasmids
pSpCas9 (BB)-2A-GFP (PX458)	Addgene	Cat# 48138; RRID: Addgene_48138
pLenti-CMV-Puro DEST (w118-1)	Addgene	Cat# 17452; RRID: Addgene_17452
pShuttle-GATEWAY-ABCC1 with STOP	GeneCopoeia	Cat# GC-Z4479
pCDNA3.1-ABCC1^K332L	Dr. Susan Cole (Queen’s University)	N/A
Software and Algorithms
Prism 7.0	GraphPad Software	N/A
Microsoft Excel	Microsoft	N/A
ImageJ 1.48v	Rasband, W.S.	https://imagej.nih.gov/ij/
FlowJo 10.1r5	Becton, Dickinson & Company	N/A