96 Chapter 4 Amino-acid restriction increases autophagic flux in mature hepatocyte-like organoids The previously reported reduction of mitochondrial proteins5, together with reduction of peroxisomal proteins and the appearance of structures resembling autophagosomes, suggests altered autophagy in the amino-acid deprived cultures. Increased appearance of autophagosomes might result either from a stabilization of autophagosomes due to an impairment in the last steps of the autophagic process, or from an increase in the formation of autophagosomes, coinciding with an elevated autophagic flux. To functionally quantify the autophagic flux in the organoids, we established a system based on expression of the GFP-LC3-RFP-LC3ΔG probe, as reported by Kaizuka et al.36 Upon autophagy activation, ATG4 cleaves the probe into equal amounts of GFP-LC3 and RFP-LC3ΔG. GFP-LC3 is then degraded in a normal way, while RFP-LC3ΔG is stable, and therefore not degraded, and works as an internal standard. Liver progenitor organoids were transduced to express the autophagic flux probe as described on Figure 2a. Two days post-transduction, stable progenitors were harvested and the cells were sorted to get an enriched population of double positives for the expression of both GFP and RFP (Figure 2b,c). Figure 2b shows the gating strategy used for the enrichment of double positive cells. From the whole population (gate A) we excluded duplets and cellular aggregates (gate B); the enriched population is depicted in gate C (Figure 2b). As expected36, amino-acid deprivation activated the autophagy flux in the organoids, as can be inferred from a reduced GFP/RFP ratio (Figure 2d, e). While after 48 hours of amino-acid restriction there was only a mild downward trend of the GFP/RFP ratio (Figure 2d upper panel, e), after 96 hours the GFP/ RFP ratio was substantially and significantly reduced, indicating a clear timedependent activation of autophagy (Figure 2d lower panel, e). Surprisingly, the autophagy inhibitor bafilomycin A1 caused only minor increase of the GFP/ RFP ratio in 48 h starved organoids, without any effect after 96 h (Figure 2d, e). Interestingly, while amino-acid restriction-induced autophagy could not be prevented with chloroquine, chemically-induced autophagy (using rapamycin) could be prevented by chloroquine co-treatment (Supplementary Figure 1). This may indicate that prolonged amino-acid-restriction induced autophagy is not sensitive to classical autophagy inhibitors such as Bafilomycin A1 (Figure 2d lower panel, e) and chloroquine (Supplementary Figure 1).
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