Measurement of SQSTM1 by flow cytometry

ABSTRACT

Macroautophagy/autophagy is a regulated cellular degradation process essential as a pro-survival mechanism and integral to the regulation of diverse cellular processes in eukaryotes. During cellular stress and nutrient sensing, SQSTM1/p62 (sequestosome 1) functions as a key receptor for selective autophagy by shuttling ubiquitinated cargoes toward autophagic degradation making it a useful marker for monitoring autophagic flux. We present a straightforward and rapid flow cytometric assay for the quantitative measurement of intracellular SQSTM1 with improved sensitivity to conventional immunoblotting and with the benefit of higher throughput and reduced requirements for starting cellular materials for adequate analysis. We demonstrate that flow cytometry is able to detect similar trends in the measurement of intracellular SQSTM1 levels following serum starvation, genetic manipulations, and bafilomycin A₁/chloroquine treatments. The assays utilizes readily available reagents and equipment without the need for transfection and utilizes standard flow cytometry equipment. In the present studies, expression of reporter proteins was applied to a range of SQSTM1 expression levels generated by genetic and chemical manipulation in both mouse as well as human cells. In combination with appropriate controls and attention to cautionary issues, this assay offers the ability to assess an important measure of autophagic capacity and flux.

Abbreviations: ATG5: autophagy related 5 ATG7: autophagy related 7 BafA: bafilomycin A1 BMDM: bone marrow-derived macrophages CQ: chloroquine EBV: Epstein-Barr Virus EDTA: ethylenediaminetetraacetic acid FBS: fetal bovine serum gMFI: geometric mean fluorescent intensity HD: healthy donor MAP1LC3/LC3/Atg8: microtubule associated protein 1 light chain 3 MedianFI: median fluorescent intensity NTC: non-target control PBMC: peripheral blood mononuclear cells RPMI: Roswell Park Memorial Institution SQSTM1/p62: sequestosome 1 WT: wild type

KEYWORDS:

• Autophagy
• Bafilomycin A₁
• chloroquine
• flow cytometry
• serum starvation
• Sqstm1/P62

Acknowledgements

We would like to thank Jon Fintzi (NIAID) for helpful discussion and biostatistical expertise and Iyadh Douagi (NIAID) for helpful discussion and flow cytometric expertise.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. The raw flow cytometric datasets used and/or analyzed during the current study are available from the corresponding author (PRW) on reasonable request.

Supplemental material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2023.2224074

Additional information

Funding

This work was supported by the Intramural Research Programs of the National Institute of Allergy and Infectious Diseases, (NIAID) (AI001123 and A0001124). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIAID or the NIH.