FK866 mediates autophagy by transcriptional-dependent and independent mechanisms. (A-B) RPMI-8226/S, MM-1S, and U266 cells were cultured with FK866 (0-10nM) for 24 to48 hours. Whole-cell lysates were subjected to immunoblotting using with the indicated antibodies. (C-D) Hierarchical clustering analysis of gene expression profiles of MM-1S cells treated with FK866 versus control. In panel C are shown autophagy-related genes (Pearson correlation 0.997 and 0.994 at 6 and 24 hours, respectively; P < .0001); in panel D MAPK-related genes (Pearson correlation .911 at 6 hours and .921 at 24 hours; P < .0001); fold change in the expression of FK866-treated cells relative to expression in untreated cells is shown by the intensity of induction (red) or suppression (green). The Gene expression data have been deposited in Gene Expression Omnibus (GEO; GSE35414) as reported in supplemental Methods. (E) U266 cells ectopically expressing a DDK-TFEB fusion protein were starved or treated with FK866 for 0 to 48 hours. Immunoblot analysis of DKK, GAPDH, and nucleolin was performed in nuclear/cytosolic extracts. (F) U266 cells were transfected with 200nM of siRNA ERK 1/2 or nontargeting siRNA. Two days later, cells were subjected to immunoblotting (top panel) or plated in 96-well plates and incubated with FK866 (0-10nM). The specific cell death was measured after 72 hours by PI staining and cytometry. A representative experiment is presented.