Autophagy : Reagent Selection Guide

Recent research shows that autophagy inhibits protein aggregation and DNA lesions in cells, leading to cellular health and extended lifespan. Here are some of the papers that show some mechanisms and factors that control autophagy for cellular homeostasis.

Autophagy is a cellular process that removes damaged organelles and proteins to maintain cellular homeostasis and function. By reducing the accumulation of toxic aggregates and dysfunctional components, autophagy helps to prevent cellular stress and inflammation, key contributors to aging. Increased autophagic activity is associated with improved proteostasis, stress resistance and increased lifespan in several model organisms. Thus, autophagy is a critical mechanism linking cellular health to healthy aging and longevity.

Point of Interest
- Mesencephalic astrocyte-derived neurotrophic factor 1 (MANF-1 regulates autophagy and lysosomal function via HLH-30/TFEB signaling to promote proteostasis and extend lifespan in C. elegans.

- MANF-1 localizes to lysosomes, modulates autophagic flux and reduces protein aggregation, thereby promoting neuronal survival and stress response.

- MANF is essential for cellular proteostasis, stress response and regulation of ageing through ER-lysosomal localisation and transcriptional signaling.

Point of Interest
- MYTHO, which is conserved across species, promotes lifespan, health and stress resistance by initiating autophagy through the WIPI2-BCAS3 scaffold.

- MYTHO deletion shortens lifespan, reduces oxidative stress resistance and disrupts autophagy, highlighting its role in healthy aging.

- MYTHO is a transcriptionally regulated autophagy initiator essential for stress resistance and longevity in C. elegans and humans.

Point of Interest
- Autophagy processes TOP1cc DNA lesions via lysosomes, enabling DNA repair and genome stability in vertebrates.

- The autophagy receptor TEX264 detects TOP1cc at replication forks and triggers p97-mediated lysosomal delivery through MRE11- and ATR-dependent pathways.

- The conserved role of selective autophagy in DNA repair supports cell survival and is clinically relevant.

Analysis of autophagic flux without transfection

DALGreen and DAPRed labeled HeLa cells were used to evaluate changes in autophagic flux induced by the lysosomal acidification inhibitor bafilomycin A1 (Baf. A1). Compared to starvation conditions, the fluorescence signals of DALGreen were decreased under inhibited conditions of autolysosome formation by the addition of Baf. A1. In contrast, the fluorescence signals of DAPRed were increased under the same conditions, indicating that Baf. A1 led to the accumulation of autophagosome.

Experimental Data

Experimental Conditions
CTRL: Normal condition, Stv.: Induction of autophagy, Stv. + Baf. A1: Inhibition of autolysosome formation
DALGreen filter set: 488 nm (Ex), 490–550 nm (Em)
DAPRed filter set: 561 nm (Ex), 565–700 nm (Em)

Procedure
1. HeLa cells were seeded (1.0 x 10⁴ cells/well) on a μ-slide 8 well plate (ibidi) and cultured overnight at 37°C in an incubator equilibrated with 95% air and 5% CO₂.
2. After washing twice with MEM containing 10% fetal bovine serum, 200 μl of DALGreen/DAPRed working solution (DALGreen: 1 µmol/l, DAPRed: 0.2 µmol/l) and the cells were incubated at 37°C for 30 minutes.
3. The supernatant was discarded, and the cells were washed twice with MEM containing 10% fetal bovine serum.
4. Samples were prepared under the following conditions.
  •　MEM containing 10% fetal bovine serum (200 µl) was added to the well, and the cells were incubated at 37°C for 2 hours 20 minutes. (Control)
  •　Amino acid-free medium (FUJIFILM Wako Pure Chemical Industries, Ltd., Catalogue code: 048-33575) (200 μl) was added to the well, and the cells were incubated at 37°C for 2 hours 20 minutes. (Starvation)
  •　Amino acid-free medium (200 μl) was added to the well, and the cells were incubated at 37°C for 2 hours. The supernatant was discarded, bafilomycin A1 working solution (10,000 times dilution, 200 μl), an inhibitor of lysosomal acidification, was added to the well, and the cells were incubated at 37°C for 20 minutes. (Inhibition of autolysosome formation)
5. The stained cells were observed under a confocal fluorescence microscope.

Products in Use
Autophagic Flux Assay Kit