Flux-Based Assay for the Identification of Autophagy Modulators for Osteoarthritis

Summary

This paper details monitoring autophagy flux to identify new molecules by cell-based imaging screening.

Abstract

Autophagy is a central mechanism to regulate homeostasis. Alterations of autophagy contribute to aging-related diseases. Phenotypic methods to identify regulators of autophagy could be used for the identification of novel therapeutics. This article describes a cell-based imaging screening workflow developed to monitor autophagic flux using LC3 as a reporter of autophagic flux (mCherry-EGFP-LC3B) in human chondrocytes. Data acquisition is performed using an automated High Content Imaging Screening System microscope. An algorithm-based automated image analysis protocol was developed and validated to identify molecules activating autophagic flux. Critical steps, explanatory notes, and improvements over current autophagy monitoring protocols are reported. Physiologically relevant phenotypic screening approaches to target hallmarks of aging can facilitate more effective drug discovery strategies for age-related musculoskeletal diseases.

Introduction

Many chronic diseases are associated with the hallmarks of aging, including defective autophagy¹. Osteoarthritis (OA) is the most prevalent joint disease and has a major impact in restricting everyday activities in the aging population, but neither preventive measures nor disease-modifying treatments are yet available².

Joint aging and OA are associated with hallmarks that define the progression of cartilage degeneration, including defective autophagy and senescence^3,4. Targeting autophagy in musculoskeletal tissues can help find ....

Protocol

1. Generation of autophagy reporter cell line of immortalized human chondrocytes

NOTE: The generation of an autophagy reporter cell line by stable transfection of pBABE-mCherry-GFP-LC3 by establishing a flow cytometry quantitative readout was described previously¹¹. Two cell lines were used in the retroviral transfection: HEK 293-T17 during the cotransfection process and T/C28a2 in the infection step.

1. Cotransfection process
   1. Prepare the growth medium for HEK 293-T17 cells: 500 mL of Eagle’s minimum essential medium (EMEM), 10% fetal bovine serum (FBS), and 1% penicillin-streptomycin (P/S)....

Results

Autophagic flux can be monitorized in cells by pharmacological modulation or by cell stress response. Inmortalized Human Chondrocytes (T/C28a2) were employed to develop an autophagy reporter cell line using LC3 as a fluorescent reporter (mCherry-EGFP-LC3B) for autophagy. Figure 1 shows the schematic workflow of the screening assay starting with the development of the autophagy reporter cell line in human chondrocytes (mCherry-EGFP-LC3-T/C28a2), to the induction or inhibition of autophagic fl.......

Discussion

Defective autophagy is an important hallmark of aging-related joint degeneration, but neither preventive nor disease-modifying treatments targeting autophagy are yet available for cartilage degeneration². Given its relevance and clinical implications, autophagy has become a target of interest for drug discovery and development, although methods to directly monitor changes in this key homeostasis mechanism has proved challenging.

Here, a cell-based phenotypic assay to de.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
100 mm cell culture plate	Corning	430167	Cell culture plate
12-well multiplate	Corning	353043	Cell culture plate
15 mL Centrifuge conical tube	Falcon-Corning	352095	Centrifuge conical tube
24-well multiplate	Corning	351147	Cell culture plate
25 cm2 Cell Culture Flask	Falcon-Corning	353014	Cell culture flask
384-well multiplate Cell Carrier	Perkin Elmer	6007550	Cell culture plate
6-well multiplate	Corning	351146	Cell culture plate
96-well multiplate	Corning	353077	Cell culture plate
Acoustic liquid handling technology	Labcyte	−	https://www.labcyte.com
Chloroquine	Sigma-Aldrich	C6628	autophagic flux inhibitor
Dimethyl Sulfoxide (DMSO)	Sigma-Aldrich, St. Louis, MO	D2650	Disolvent
Dulbecco's Modified Eagle's Medium (DMEM)	Lonza, Basel, Switzerland	BE-604F	T/C28a2 growth medium
Eagle's Minimum Essential Medium (EMEM)	ATCC	30–2003	HEK 293-T17 growth medium
FACScalibur cytometer	Becton Dickinson, CA	−	https://www.bdbiosciences.com/en-eu
Fetal Bovine Serum (FBS)	Sigma-Aldrich, St. Louis, MO	F9665	HEK 293-T17 serum
Fetal Calf Serum (FCS)	Gibco by Life Technologies, CA	26010–074	Serum
FuGene	Promega	E2691	A nonliposomal mixture of lipids as a plasmid delivery method to create autophagy reported cell line
Handheld electronic 384 channel pipette	Integra	−	https://www.integra-biosciences.com/united-states/en/electronic-pipettes/viaflo-96384#downloads
Hank's Balanced Salt Solution (HBSS)	Sigma-Aldrich	H6648	Buffer
HEK 293-T17	ATCC	CRL-11268	Kidney cell line. Cells were used to facilitate retroviral packaging
High Content Screening System	Perkin Elmer	−	https://www.perkinelmer.com/es/product/operetta-cls-system-hh16000000
Hoechst 33342	Thermo Fisher Scientific	62249	DNA staining
Image Analysis Software	Perkin Elmer	−	https://www.perkinelmer.com/es/product/harmony-4-9-office-license-hh17000010
Liquid Handler workstation	Perkin Elmer	−	https://www.perkinelmer.com/es/category/janus-liquid-handler-workstations
Microplate washer robot	Biotek	−	https://go.biotek.com/405tradein
Opti-MEM® (1x)	Thermo Fisher Scientific	11058	Transfection medium
Paraformaldehyde (PFA)	Sigma-Aldrich	158127	Fixer
pBABE-puro mCherry-EGFP-LC3B	Addgene, Cambridge, MA	22418	Plasmid
pCL-Eco	Addgene, Cambridge, MA	12371	Plasmid
Penicillin-Streptomycin (P/S)	Sigma-Aldrich	P0781	Antibiotic
Phosphate-buffered saline (PBS)	MP Biomedicals	2810305	Buffer
Puromycin	Sigma-Aldrich, St. Louis, MO	P8833	Antibiotic
Rapamycin	Calbiochem, Germany	5053210	autophagic flux activator
Software CellQuestPro	Becton Dickinson	−	https://www.bdbiosciences.com/en-eu
Syringe filters 0.45 μm	Corning	CLS431220	Sterile filter
T/C28a2	−	−	human chondrocytes cell line
Trypsin	Gibco by Life Technologies, CA	15400054	Trypsin used with T/C28a2 cells
Trypsin	Sigma-Aldrich, St. Louis, MO	SM-2002-C	Trypsin used with HEK 293-T17 cells
VSV.G	Addgene, Cambridge, MA	14888	Plasmid