Figure 1. Cell-based assay for the identification of chemical modulators of autophagy and identification of amiodarone, niclosamide, perhexiline and rottlerin as modulators of autophagy.

(a) Schematic representation of the EGFP-LC3 puncta formation assay used to screen for chemical modulators of autophagy. (b) Punctate EGFP-LC3 accumulation upon treatment with rapamycin or nutrient deprivation. MCF-7 cells stably expressing EGFP-LC3 were incubated without or with 20 nM rapamycin in complete cell culture medium or in medium lacking serum and amino acids for 4 h. EGFP-LC3 punctate staining was visualised by automated microscopy as described in Materials and Methods. (c) Chemical structures of perhexiline, niclosamide, amiodarone and rottlerin. (d) Quantitation of punctate EGFP-LC3 accumulation upon incubation with the active chemicals. MCF-7 cells stably expressing EGFP-LC3 were incubated for 4 h with different concentrations of the chemicals and EGFP-LC3 puntacte staining was quantitated using the automated microscopy assay.