KEY RESOURCES TABLE

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
HA Tag Mouse Monoclonal Antibody (2-2.2.14)	Invitrogen	Cat#26183
β-Actin Rabbit Monoclonal Antibody	LI-COR	Cat# P/N 926-42210
HSF1 Antibody #4356	Cell Signaling	Cat# 4356T
Anti-Iba1, Rabbit (for Immunocytochemistry)	FujiFilm Wako Chemicals	Cat# 019-19741
Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488	Invitrogen	Cat# A-11008
IRDye® 680RD Goat anti-Mouse IgG Secondary Antibody	LI-COR	Cat# P/N: 926-68070
IRDye® 800CW Goat anti-Rabbit IgG Secondary Antibody	LI-COR	Cat# P/N: 926-32211
Bacterial and Virus Strains
Lentivirus: R12Y/G67S/Y100I ecDHFR.YFP.HA	This paper	N/A
Lentivirus: I5F/R12Y/G67S/Y100I ecDHFR.YFP.HA	This paper	N/A
Lentivirus: R12Y/M16L/G67S/Y100I ecDHFR.YFP.HA	This paper	N/A
Lentivirus: R12Y/F31L/G67S/Y100I ecDHFR.YFP.HA	This paper	N/A
Lentivirus: R12Y/G67S/I94L/Y100I ecDHFR.YFP.HA	This paper	N/A
Lentivirus: ecDHFR.dn-cHSF1	This paper	N/A
AAV Virus: AAV2/2 MAX NanoLuc 2A ecDHFR FLuc	Packaged by UNC Viral Vector Core	N/A
AAV Virus: AAV2/8 NanoLuc 2A ecDHFR.Fluc	Packaged by UNC Viral Vector Core	N/A
Bacteria: wild-type S. aureus	Marrafini Laboratory, Rockefeller University	RN4220
Bacteria: wild-type E. coli	Toprak Laboratory, UT Southwestern	BW25113
Chemicals, Peptides, and Recombinant Proteins
Dimethyl Sulfoxide (DMSO)	Fisher Scientific	Cat# BP231-100
Carbowax™ PEG 400 (NF)	Fisher Scientific	Cat# P167-1
Tween™ 80	Fisher Scientific	Cat# BP338-500
Tween™ 20	Fisher Scientific	Cat# BP337-100
Cremophor EL	Millipore Sigma	Cat# 238470
Dextrose Anhydrous (Crystalline Granules/Molecular Biology)	Fisher Scientific	Cat# BP350-500
HBSS	Sigma	Cat# H6648
D-Luciferin Sodium Salt	Gold Biotechnology	Cat# LUCNA
0.9% Sodium Chloride Injection, USP	Hospira	N/A
Ponceau S solution	Sigma	Cat# P7170
2-aminoquinazoline	Sigma	Cat# CDS008332
2-amino-3H-quinazolin-4-one	Sigma	Cat# CDS020257
2,4-diaminopyrimidine	Sigma	Cat# 68231
2,4-diamonopyrimidine-5-carbaldehyde	Sigma	Cat# CDS013913
Dihydrofolic acid	Sigma	Cat# D7006
pemetrexed	Tocris Bioscience	Cat# 6185
Proguanil HCl	Acros Organics	Cat# 637-32-1
raltitrexed	Apex Bioscience	Cat# B1476
RG3039	Butchbach Laboratory, University of Delaware	N/A
D156844	Butchbach Laboratory, University of Delaware	N/A
Trimethoprim	Sigma	Cat# T7883
Methotrexate	Fisher Scientific	Cat# BP2665
Triamterene	Selleck Chemicals	Cat# S4080
Ormetoprim, 98%	J&K	Cat# 403370
Pyrimethamine	Tocris Bioscience	Cat# 3918
SW000198-2	ChemDiv	Cat# 0075-0058
Pralatrexate	Apex Bio	Cat# A4350
Diaveridine	Santa Cruz	Cat# SC-205646
Aminopterin	Sigma	Cat# A3411
Cycloguanil	Cayman Chemical	Cat# 16861
2,4-diaminoquinazoline	Alfa Aesar	Cat# L13783
WR99210	Sigma	Cat# W1770
GW2580	Apex Bio	Cat# A1655
TC-P 262	Tocris Bioscience	Cat# 4386
Epiblastin A	Sigma	Cat# SML1647
TG100-115	Selleck Chemicals	Cat# S1352
7,8-DIETHOXY-QUINAZOLINE-2,4-DIAMINE	Sigma	Cat# R396540
HZ-1157	AChemBlock	Cat# O33918
Critical Commercial Assays
Pierce™ BCA Protein Assay Kit	ThermoFisher Scientific	Cat# 23227
Dihydrofolate Reductase Assay Kit	Sigma	Cat# CS0340
Maxi Prep Plus Kit	Qiagen	Cat# 12943
Deposited Data
E. coli DHFR substrate complex with dihydrofolate	Cao et al., 2018	PDB ID: 6CXK
E. coli DHFR complex with methotrexate	Sawaya and Kraut, 1997	PDB ID: 1RA3
M. tuberculosis DHFR in complex with cycloguanil	Dias et al., 2014	PDB ID: 4KNE
Experimental Models: Cell Lines
Human: HEK-293A	Life Technologies	Cat# R70507
Human: HeLa Tet-On	Takara	Cat# 631183
Human: ARPE-19	American Type Culture Collection	Cat# CRL-2302
ARPE-19 R12Y/G67S/Y100I ecDHFR.YFP.HA	This paper	N/A
ARPE-19 I5F/R12Y/G67S/Y100I ecDHFR.YFP.HA	This paper	N/A
ARPE-19 R12Y/M16L/G67S/Y100I ecDHFR.YFP.HA	This paper	N/A
ARPE-19 R12Y/F31L/G67S/Y100I ecDHFR.YFP.HA	This paper	N/A
ARPE-19 R12Y/G67S/I94L/Y100I ecDHFR.YFP.HA	This paper	N/A
HeLa R12Y/G67S/Y100I ecDHFR.YFP.HA	This paper	N/A
HeLa ecDHFR.dn-cHSF1	This paper	N/A
Experimental Models: Organisms/Strains
Mouse: Wild Type Balb/c	Jackson Laboratories, private stock courtesy of Lihua Marmorstein	N/A
Oligonucleotides
Primer: I5F ecDHFR Forward 5’ GATCAGTCTGTTTGCGGCGTTAG ‘3	Sigma	N/A
Primer: I5F ecDHFR Reverse 5’ ATGGTGGCGGATCCAGTC ‘3	Sigma	N/A
Primer: I94L ecDHFR Forward 5’ AATCATGGTGCTTGGCGGCGG ‘3	Sigma	N/A
Primer: I94L ecDHFR Reverse 5’ TCTGGTACGTCACCACAC ‘3	Sigma	N/A
Primer: F31L ecDHFR Forward 5’ TCGCCTGGTTAAAACGCAACAC ‘3	Sigma	N/A
Primer: F31L ecDHFR Reverse 5’ GATCGGCAGGCAGGTTCC ‘3	Sigma	N/A
Primer: M16L ecDHFR Forward 5’ CGTTATCGGCCTGGAAAACGC ‘3	Sigma	N/A
Primer: M16L ecDHFR Reverse 5’ TAATCTACCGCTAACGCC ‘3	Sigma	N/A
Primer: CMV Forward Sequencing Primer 5’ CCAAGTACGCCCCCTATTGA ‘3	Sigma	N/A
Recombinant DNA
pAAV2/2 MAX (7m8, QuadYF)	Reid et al., 2017	N/A
pAAV2/8	Zhu Laboratory, UT Southwestern	N/A
pTR smCBA Nluc 2A ecDHFR Fluc	Peng et al., 2019	N/A
pLenti Puro R12Y/G67S/Y100I ecDHFR.YFP.HA	This paper	N/A
pLenti Puro I5F/R12Y/G67S/Y100I ecDHFR.YFP.HA	This paper	N/A
pLenti Puro R12Y/M16L/G67S/Y100I ecDHFR.YFP.HA	This paper	N/A
pLenti Puro R12Y/F31L/G67S/Y100I ecDHFR.YFP.HA	This paper	N/A
pLenti Puro R12Y/G67S/I94L/Y100I ecDHFR.YFP.HA	This paper	N/A
pLenti Puro ecDHFR-dn.cHSF1	Moore et al., 2016	N/A
Software and Algorithms
GraphPad Prism	GraphPad Software, Inc	https://www.graphpad.com/scientific-software/prism/
CellProfiler 3.1.8	Broad Institute	https://cellprofiler.org/releases/
Protein Preparation Wizard	Schrodinger LLC	https://www.schrodinger.com/protein-preparation-wizard
Maestro v11.9.011	Schrodinger, LLC	https://www.schrodinger.com/maestro
SnapGene	GSL Biotech	https://www.snapgene.com/
Other
National Cancer Institute (NCI) preclinical experimental collection	NCI	N/A
Prestwick Chemical Library	Prestwick Chemical	http://www.prestwickchemical.com/libraries-screening-lib-pcl.html
ad hoc portion of University of Texas Southwestern 300k collection	UT Southwestern	N/A
eMolecules Chemical Database	eMolecules, Inc.	https://www.emolecules.com/
MolPort Chemical Database	MolPort	https://www.molport.com/shop/index