Development of small fluorescent probes for the analysis of autophagy kinetics

. 2023 Jun 28;26(7):107218. doi: 10.1016/j.isci.2023.107218

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies

Mouse monoclonal anti-LC3 (1:1000)	Nano Tools	Cat# 0231-100/LC-5F10; RRID: AB_2722733
Rabbit polyclonal anti-SQSTM1/p62 (1:1000)	MBL	Cat# PM045; RRID: AB_1279301
Mouse monoclonal anti-TUBA (1:2500)	Sigma-Aldrich	Cat# T9026; RRID: AB_477593
Rabbit monoclonal anti-Phospho4E-BP1(Thr37/46) (1:1000)	Cell Signaling	Cat# 2855; RRID: AB_2097841
Rabbit monoclonal anti-4E-BP1 (1:1000)	Cell Signaling	Cat# 9644; RRID: AB_2097841
Rabbit polyclonal anti-Atg5 (1:1000)	Novus	Cat# NB110-53818; RRID: AB_828587

Chemicals, peptides, and recombinant proteins

Amylamine	TCI	Cat# A0445
Perylene-3,4-dicarboxylic anhydride	Iwashita et al.⁸	N/A
2-(Boc-amino)ethyl bromide	TCI	Cat# B2289
Baffilomycin A1	Focus Biomolecules	Cat# 10-2060
DMSO	Wako	Cat# 041-29351
Hanks' Balanced Salt Solution	Nacalai	Cat# 09735-75
DALGreen	Dojindo	Cat# D675
DAPGreen	Dojindo	Cat# D676
DAPRed	Dojindo	Cat# D677
Rapamycin	Santa Cruz Biotechnology	Cat# SC-3504
Chloroquine diphosphate	Nacalai	Cat# 08660-62
Etoposide	Sigma-Aldrich	Cat# E1383
SBI-0206965	Sigma-Aldrich	Cat# SML1540
3-Methyladenine	Sigma-Aldrich	Cat# M9281
LysoTracker Red DND-99	Thermo Fisher Scientific	Cat# L7528
LysoTracker Deep Red	Thermo Fisher Scientific	Cat# L12492
MitoTracker Deep Red FM	Thermo Fisher Scientific	Cat# M22426
MitoTracker Green FM	Thermo Fisher Scientific	Cat# M7514
ER-Tracker Red	Thermo Fisher Scientific	Cat# E34250
SNAP-Cell 430	New England Biolabs	Cat# S9109S
HaloTag SaraFluor650T ligand	Goryo Chemical	Cat# A308-01
EYPC	Wako	Cat# 124-05031
PI3P	Echelon	Cat# P-3016
PlasMem Bright Red	Dojindo	Cat# 346-09771
Wortmannin	Merck Calbiochem	Cat# 681675
Dulbecco’s modified Eagle’s medium	Nacalai	Cat# 08458-45
MEM vitamin solution	Gibco	Cat# 11120-052
L-glutamine	Nacalai	Cat# 16948-04
sodium pyruvate	Nacalai	Cat# 06977-34
nonessential amino acids	Gibco	Cat# 11140-050
HEPES	Nacalai	Cat# 17557-94
2-mercaptoethanol	Nacalai	Cat# 21438-82
penicillin-streptomycin mixed solution	Nacalai	Cat# 26253-84
fetal bovine serum	Biowest	Cat# S1810-500
paraformaldehyde	Nacalai	Cat# 26126-25
glutaraldehyde	TAAB	Cat# 3041
OsO4	TAAB	Cat# 300
Lead stain solution	Sigma-Aldrich	Cat# 18-0875
Tricaine	Sigma-Aldrich	Cat# E10521

Critical commercial assays

CytoID	Enzo Life Science	Cat# ENZ-51031-K200
Neon transfection system	Thermo Fisher Scientific	Cat# MPK1096
AMAXA Cell Line Nucleofector Kit V	Lonza	Cat# VCA-1003
Lipid Snoopers, PIPs	Avanti	Cat# 330500

Experimental models: Cell lines

HeLa cell (Wild type)	Iwashita et al.⁸	N/A
Atg8 hexa-KO HeLa cell	Nguyen et al.²⁰	N/A
Mouse embryonic fibroblast (WT MEF)	Shimizu et al.²⁴	N/A
Atg5-deficient MEF	Nishida et al.⁹	N/A
Atg9-deficient MEF	Saitoh et al.²⁵	N/A
Ulk1/2-deficient MEF	McAlpine et al.²⁶	N/A

Experimental models: Organisms/strains

Zebrafish (Tüpfel long fin)	Asaoka et al.²⁷	ZFIN: ZDB-GENO-9900623-2

Oligonucleotides

Wipi2 modification primer F1: 5′-GTACAAGGGATCCATGAACCTGGCGAGCCAGAGC-3′	This paper	N/A
Wipi2 modification primer F2: 5′-TAGATCCATGGTGAGCAAGGGCGAGGAGGATAAC-3′	This paper	N/A
Wipi2 modification primer R1: 5′-CTCACCATGGATCTAATTCCGGCGCCTAGAGAAG-3′	This paper	N/A
Wipi2 modification primer R2: 5′-ATGGATCCCTTGTACAGCTCGTCCATGCCGCCGGTG-3′	This paper	N/A
GABARAP modification primer F: 5′-GCCAGATCTATGAAGTTCGTGTACAAAGAAGAGCATC-3′	This paper	N/A
GABARAP modification primer R: 5′-GCCCTCGAGTCACAGACCGTAGACACTTTCGTCACTG-3′	This paper	N/A
Cytb5^104-¹³⁴ modification primer F1: 5′-taatagatctggctccggaggaggaaagccaatcc-3′	This paper	N/A
Cytb5^104-¹³⁴ modification primer R1: 5′-gagccagatctattaagtttgtgccccagtttgc-3′	This paper	N/A
Cytb5^104-¹³⁴ modification primer F2: 5′-gactgagaattctgcagatatccagcacagtggc-3′	This paper	N/A
Cytb5^104-¹³⁴ modification primer R2: 5′-gcagaattcTCAGTCCTCGGCCATATACAGCCTG-3′	This paper	N/A
STX17 truncation primer F: 5′-CTGCCAGCGGATCCGTTAACTCGCGAACGCGT-3′	This paper	N/A
STX17 truncation primer R: 5′-CGGATCCGCTGGCAGCTCTGCCTGTGGCAGGTG-3′	This paper	N/A
ST6GAL1 cloning primer F: 5′-CTTAAGCTTGAAGATGATTCACACCAACCTGAAGAAAAAG-3′	This paper	N/A
ST6GAL1 cloning primer R: 5′-GTGGATCCTTGCAGTGAATGGTCCGGAAGCCAGGC-3′	This paper	N/A
Tom20 cloning primer F: 5′-CTCGGATCCATGGTGGGCCGGAACAGCG-3′	This paper	N/A
Tom20 cloning primer R: 5′-CATGAATTCTTCCACATCATCCTCACCC-3′	This paper	N/A
Atg5MO: 5′-CCTTGTCATCTGCCATTATCATCGT-3′	Varga et al.²³; GeneTools	N/A
custom control oligo (5′-CCTTCTCAGCTCCCATAATCTTCGT-3′	This paper; GeneTools	N/A

Recombinant DNA

pmCherry-WIPI2	This paper	N/A
pEGFP-WIPI2	This paper	N/A
pEGFP-LC3	This paper	N/A
pmCherry-LC3	This paper	N/A
pmCherry-EGFP-LC3	This paper	N/A
pTagRFP-LC3	Yamaguchi et al.¹¹	N/A
pGABARAP-Venus	This paper	N/A
pGABARAP-TagRFP	This paper	N/A
pLamp1-Venus	This paper	N/A
pSNAP f vector	New England Bio Labs	Cat# N9183S
pLamp1-SNAPtag	This paper	N/A
pLamp1-TagRFP	Torii et al.²⁸	N/A
pEGFP-Cytb5^104-134	Tashiro et al.²⁹	N/A
pTagRFP-Cytb5^104-134	This paper	N/A
pTom20-EGFP	This paper	N/A
pTom20-mRFP	This paper	N/A
pEGFP-STX17TM	This paper	N/A
pST6GAL1-EGFP	This paper	N/A
pST6GAL1-TagRFP	This paper	N/A
pEGFP-Rab9	Nishida et al.⁹	N/A
pAtg9-EGFP	Takahashi et al.³⁰	N/A
pHaloTag-LC3	Promega	Cat# FHC05917

Software and algorithms

ImageJ (Fiji)	Fiji project	https://imagej.net/
GraphPad Prism 5	GraphPad Software	https://www.graphpad.com/
SnapGene4.1.6	GSL Biotech	http://www.snapgene.com/
ApE-A plasmid Editor v2.0.47	EM. Jorgensen laboratory (University of Utah)	https://jorgensen.biology.utah.edu/
ZEN2.1	Carl Zeiss	https://www.zeiss.com/
BZ-H4C	Keyence	https://www.keyence.com/
THUNDER 3D Cell Culture Imager	Leica	https://www.leica-microsystems.com/
FACSDiva	BD	https://www.bdbiosciences.com/