Autophagy LC3 HiBiT Reporter Assay System Demonstrates mTORC1 Regulation of Autophagic Flux

Autophagy is an evolutionarily conserved intracellular degradation process that is implicated in many physiological and pathological conditions, such as cancer (1).  When autophagy is induced, an isolation membrane is formed that encloses part of the cytoplasm and forms a double-membraned vesicle (autophagosome). The autophagosome outer membrane fuses with a lysosome to form the autolysosome, degrading the engulfed organelle components along with the inner autophagosome membrane.

Mammalian LC3B is an autophagosome marker that is processed by Atg4 at its C-terminus to become LC3-I. LC3-I is mostly cytosolic, but is lipidated through phosphatidylethanolamine (PE) conjugation to form LC3-II that localizes at both the outer and inner membranes of the autophagosome. Upon autophagosome fusion with the lysosome, the inner-membrane bound LC3-II is degraded by lysosomal proteases.

There are several methods for monitoring autophagosome formation (e.g., electron microscopy morphological studies, imaging of GFP-LC3 puncta formation, Western blot of LC3I-II conversion) and autophagic flux (e.g., imaging of the mRFP-GFP-LC3 reporter, Western blot of GFP-LC3 cleavage, LC3I/II ratio or total levels of selective autophagy substrates like p62, and long-lived protein degradation) (2, 3). However, each method has its limitations. The results obtained should be interpreted with caution, and more than one method should be used to validate autophagic activity. For example, using the number of autophagosomes determined by GFP-LC3 localization is a common method for measuring autophagy activity. However, the results might be misleading. This is because both induction and inhibition of autophagy can lead to increased autophagosome accumulation.

The Autophagy LC3 HiBiT Reporter Assay System measures autophagic flux by monitoring total levels of the LC3-based reporter (Figure 1). Cells are stably transfected with a plasmid encoding human LC3B protein tagged with HiBiT peptide and an intervening spacer. Upon addition of the detection reagent containing Large BiT protein (LgBiT: inactive luciferase subunit), LgBiT binds with high affinity to HiBiT to generate an active luciferase enzyme. Addition of the luciferase substrate produces bioluminescence that correlates with total LC3-based reporter levels.

We tested the effects of known autophagy inducers on U2OS Autophagy LC3 HiBiT Reporter Cells by inhibiting major regulators of autophagy, mTORC1 and mTORC2. When mTORC1 is inhibited by starvation or treatment with ATP-competitor mTOR inhibitors such as Torin 1, autophagy is induced (4). Pharmacological treatments often cause off-target or unspecific effects. In this case, Torin 1 can inhibit both mTORC1 (containing mTOR, Raptor and mLST8) and mTORC2 (containing mTOR, Rictor and mLST8) (5).

To determine the effect of mTORC1 and mTORC2 on autophagic activity, we performed genetic studies by knocking-down Raptor or Rictor—essential for mTORC1 or mTORC2 activity, respectively (Figure 2A). Efficient Raptor and Rictor knockdown was confirmed by Western blot (Figure 2B). As expected, Raptor knockdown largely reduced HiBiT-tagged LC3 reporter levels compared to the non-targeting control. However, Rictor knockdown did not show a significant difference compared to the control-treated cells, indicating that mTORC2 has limited or no effect on autophagy activity in the cell system analyzed. 

One limitation of the assay system is the possibility that reporter signal might be impacted by changes in cell number, depending on time and toxicity of treatments. Several options can be considered to mitigate this risk, such as performing fluorescent nuclei labeling methods in the same wells used to obtain reporter luminescence.

This Autophagy LC3 HiBiT Reporter Assay System is very sensitive and allows a large number of conditions to be tested simultaneously, making it useful for large-scale compound screening. With this assay, changes in autophagic flux can easily be determined in any cell line stably transfected with the Autophagy LC3 HiBiT Reporter Vector. It is also useful for distinguishing autophagy stimulation from late-phase pathway inhibition.

Antibodies and Reagents

Torin 1 was purchased from BioCat and Bafilomycin A1 from AlfaAesa. Antibodies were purchased from the following sources and used at the indicated dilution: Cell Signaling: Raptor (2280, 1:1000), Rictor (2114, 1:1000); Progen: p62 (GP62-C, 1:2000 dilution); Novus Biologicals: LC3B (NB100-2220, 1:2000); Sigma: Actin (A0483, 1:10,000).

Cell Culture

U2OS Autophagy LC3 HiBiT Reporter Cell Line (Promega) was maintained in McCoy’s 5A media containing 10% FBS, 1% P/S, ʟ-Glutamine and 250 μg/ml G418 (Gibco).

siRNA Transfection

siRNA transfections were performed using Lipofectamine RNAiMax (Invitrogen) according to the manufacturer’s instructions. Human Dicer-siRNA for Raptor, Rictor and non-targeting control were obtained from Integrated DNA Technologies (IDT).

Cell Lysates and Western Blot Analyses

Cells were washed in ice-cold PBS and lysed in M-PER lysis buffer (Thermo Fisher Scientific) containing protease inhibitors (Complete Mini; Roche), 5mM NaF, 1mM Na3VO4 and phosphatase inhibitors cocktail (PhosSTOP; Roche) for 10 minutes at 4°C. Lysates were cleared by centrifugation at 16,000 × g for 10 minutes. Cleared lysates were diluted in 4Χ Roti Load loading buffer (Roth-Chemie GmbH) and denatured at 95°C for 10 minutes. Protein samples were loaded on a 5–20% gradient gel (Bio-Rad), transferred to PVDF membranes (Millipore) and incubated with the indicated antibodies.

Autophagy LC3 HiBiT Reporter Assays

Cells were seeded (8,000 cells/well) in white 96 well plates (Corning). Twenty-four hours later, cells were washed twice with PBS and media was changed to 50μl of normal media or starvation (HBSS) media containing the different treatments and corresponding controls for 3 hours. Autophagy LC3 HiBiT Reporter levels were measured using the Nano-Glo® HiBiT Lytic Reagent (Promega) according to manufacturer’s instructions.

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