Autophagy : Reagent Selection Guide

Science Note

How does cancer exploit the lysosome and autophagy? 

Autophagy is a cellular process that involves the degradation and recycling of cellular components through the formation of autophagosomes, which subsequently fuse with lysosomes for content degradation. In the context of cancer, autophagy plays a dual role; it can suppress tumor initiation by eliminating damaged organelles and proteins, but can also promote tumor survival and growth under metabolic stress by providing nutrients through the recycling of cellular components.
Carnosine regulation of intracellular pH homeostasis promotes lysosome-dependent tumor immunoevasion
Click here for the original article: Ronghui Yan, et. al., Nature Immunology, 2024.

Point of Interest
- Carnosine synthase, CARNS2, promotes carnosine synthesis under hypoxia.

- Carnosine controls lysosomal subcellular distribution, acidification, and activity by maintaining intracellular pH homeostasis.

- By maintaining lysosomal activity, carnosine facilitates NFX1 degradation, which triggers galectin-9 and T-cell-mediated immune escape and tumorigenesis.

Response to Bruton’s tyrosine kinase inhibitors in aggressive lymphomas linked to chronic selective autophagy
Click here for the original article: James D. Phelan​, et. al., Cancer Cell, 2024.

Point of Interest

-Chronic selective autophagy suppresses NF-κB signaling in MCD-type Diffuse large B cell lymphoma (DLBCL).

-MCD-type DLBCL develops genetic and epigenetic alterations that attenuate selective autophagy.

-Disruption of selective autophagy leads to the accumulation of ubiquitinated MYD88L265P.

-BTK and mTOR inhibitors interfere with the My-T-BCR pathway by enhancing the autophagic degradation of MYD88L265P.

In vivo CRISPR knockout screen identifies p47 as a suppressor of HER2+ breast cancer metastasis by regulating NEMO trafficking and autophagy flux
Click here for the original article: Mingang Hao, et. al., Cell Reports, 2024.

Point of Interest

-An in vivo CRISPR screen identifies p47 as a suppressor of HER2+ breast cancer metastasis.

-Depletion of p47 reduces NEMO endosomal trafficking and enhances NF-κB signaling.

-Ablation of p47 impairs lysosomal repair and autophagic flux, leading to increased metastasis.

-Lower expression of p47 correlates with increased metastasis in human breast cancer.
Related Techniques
First-time autophagy research Autophagic Flux Assay Kit
Autophagy detection dyes for imaging DAPRed (Autophagosome detection), DALGreen (Autolysosome detection)
Autophagy detection for Flow cytometry / plate assay DAPGreen (Autophagosome detection)
Mitophagy detection dye Mitophagy Detection Kit and Mtphagy Dye
Lysosomal pH and mass detection Lysosomal Acidic pH Detection Kit-Green/Red and Green/Deep Red 
Accurate detection of endocytosis by pH changes ECGreen-Endocytosis Detection
Glycolysis/Oxidative phosphorylation Assay Glycolysis/OXPHOS Assay Kit
Related Applications

Analysis of autophagic flux without transfection

Detection Principle

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

Autophagic Pathway and Reagent Selection Guide

Autophagy is a degradation process of cytoplasmic dysfunctional proteins and organelles. In this process, an isolation membrane composed of double membrane appear in cytosol, expands gradually, enfold with the aggregated proteins and damaged organelles, and close to form autophagosomes. The autophagosomes are fused with lysosomes to form autolysosomes in which are acidic environment. The contents in autolysosomes are decomposed by digestive enzymes in lysosomes. Since this cellular function is said to be related to aging as well as neurodegenerative diseases such as Parkinson’s disease, a simple autophagy detection method is being required.

 

  Small Fluorescent Molecules Fluorescent Protein
DAPGreen  DAPRed  DALGreen MDC, Cyto-ID GFP-LC3 RFP-LC3 mRFP-GFP-LC3
Autophagosome ✓ ✓ - ✓ ✓ ✓ ✓
Autolysosome ✓ ✓ ✓ ✓ - ✓ ✓

Application Data

Autophagy-lysosomal pathway

Tracing autophagosome to autolysosome in live cells

Nampt inhibitor, FK866 inhibits the progress of autophagosome to autolysosome by lysosomal deacidification. A recent finding shows that the dysfunctional condition of nicotinamide adenine dinucleotide (NAD+) biosynthetic enzyme, Nampt induces lysosomal deacidification1). In this section, we tried to determine how NAD+ depletion-induced lysosomal deacidification affects the autophagy-lysosomal pathway.

1) Mikako Yagi, et. al., EMBO J., 40(8), e105268 (2021)

To confirm the effect of the Nampt inhibitor, FK866, on lysosomal acidification, HeLa cells were first labeled by the lysosomal pH detection dye pHLys Red. The cells were then treated with FK866, and lysosomal acidification inhibitor Bafilomycin A1 was used as a positive control. FK866 and Bafilomycin A1-treatment each decreased the fluorescent pHLys Red signal, indicating lysosome neutralization.

We next determined how FK866-induced lysosomal deacidification affects the autophagy-lysosomal pathway. After staining with DAPGreen/DAPRed (for detecting autophagosome), or DALGreen (for detecting autolysosome), HeLa cells were starved in HBSS incubation and then treated with FK866 or Bafilomycin A1. Under the starvation condition, the fluorescent signals from all dyes increased, indicating the proceeding autophagy-lysosomal pathway. On the other hand, only DALGreen's signals were decreased in FK866 and Bafilomycin A1 treated cells with starvation conditions. These results clearly suggested that FK866 inhibits the autophagy-lysosomal pathway by NAD+ depletion-induced lysosomal deacidification.

Time-lapse imaging of autophagy with DALGreen

For more details, click application data

H. Iwashita, H. T. Sakurai, N. Nagahora, M. Ishiyama, K. Shioji, K. Sasamoto, K. Okuma, S. Shimizu, and Y. Ueno, “Small fluorescent molecules for monitoring autophagic flux“, FEBS Lett., 2018, 592, (4), 559–567.

Some data mentioned on this page is provided by the reference above.

 

 

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