By Christina Towers, PhD.
Autophagy is highly conserved and tightly regulated process that all cell types use to recycle nutrients, particularly in the instance of stress1. As a result, even small changes in signaling pathways, gene expression, or drug treatments can alter autophagy. If you stumbled into the field of autophagy and hypothesized a change in autophagy with your favorite cell manipulation of choice, you probably read a handful of publications and concluded that you should assay LC3 levels to test this hypothesis. In fact, LC3 expression can be a good readout for autophagy, but researchers should be cautious when interpreting and quantifying these results2.
Key points to keep in mind when assaying for LC3
The most common way to measure LC3 is via western blot, and there are clean and quantifiable antibodies for this purpose. The lipidated form of LC3 (LC3-II, the smaller band on a gel) is the active form that is incorporated into autophagosomes destined for lysosomal degradation. Any changes in LC3-II cannot be interpreted without addition of a lysosomal inhibitor to monitor autophagic flux. Increased LC3-II after any treatment can be indicative of increased autophagosome formation, resulting in increased autophagic degradation, or on the contrary could also be the result of decreased autophagic turnover, resulting in a buildup of LC3-II labeled autophagosomes. Therefore, in order to correctly interpret LC3-II protein expression, each condition should also be treated with a lysosomal inhibitor (Bafilomycin A1 or Chloroquine) for 2-4 hours.
LC3B Knockout Validation: LC3B Antibody [NB100-2220] - Western blot shows lysates of HeLa human cervical epithelial carcinoma parental cell line and LC3B knockout HeLa cell line (KO) untreated (-) or treated (+) with 50 uM Chloroquine for 18 hours. Polyvinylidene difluoride (PVDF) membrane was probed with 0.5 ug/mL of Rabbit Anti-LC3B Monoclonal Antibody [NB100-2220] followed by HRP-conjugated Anti-Rabbit IgG Secondary Antibody [HAF008]. A specific band was detected for LC3B at approximately 15 kDa in the parental HeLa cell line, but is not detectable in the knockout HeLa cell line. GAPDH is shown as a loading control. This experiment was conducted under reducing conditions.
If the LC3-II increase seen at baseline (without lysosomal inhibitors) is maintained with lysosomal inhibition, the conclusion stands that autophagy is increased with treatment. However, if the initial observation of increased LC3-II is lost with treatment of Bafilomycin A1 or Chloroquine, it is very likely that the treatment in question actually causes a decrease in autophagic flux. Moreover, because LC3 is also regulated transcriptionally, LC3-II levels should never be normalized to LC3-I but instead to an endogenous loading control like Beta-Actin.
Additional tools have also been developed that help decipher autophagic flux, including LC3-mCherry-GFP tandem constructs, whereby the GFP signal is quenched at low pH allowing for the ratio of mCherry to GFP (assessed by either flow cytometry or confocal microscopy) to determine autophagic flux3. To accurately assess LC3-mediated autophagy, additional experiments should also be conducted that investigate the turnover of canonical autophagic substrates like p62; quantitative antibodies exist for this purpose. A change in LC3-II protein expression at baseline is just the start of the journey towards implicating autophagy in any system, and the correct flux experiments must follow.
Learn More About Autophagy And LC3
Christina Towers, PhD
University of Colorado (AMC)
Dr. Towers studies the roles of autophagy, apoptosis and cell death in cancer.
- Towers, C. G. & Thorburn, A. Therapeutic Targeting of Autophagy. EBioMedicine 14, 15-23, doi:10.1016/j.ebiom.2016.10.034 (2016).
- Klionsky, D. J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition). Autophagy 12, 1-222, doi:10.1080/15548627.2015.1100356 (2016).
- Gump, J. M. & Thorburn, A. Sorting cells for basal and induced autophagic flux by quantitative ratiometric flow cytometry. Autophagy 10, 1327-1334, doi:10.4161/auto.29394 (2014).