Autophagy : Reagent Selection Guide

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.

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 MYD^88L265P.

-BTK and mTOR inhibitors interfere with the My-T-BCR pathway by enhancing the autophagic degradation of MYD^88L265P.

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.

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