Figure 1. Isolation of a Series of Small Molecule Inhibitors of Autophagy.

(A) The structure of MBCQ.

(B) MBCQ reduced the spot numbers (a), spot size (b), and spot intensity (c) of LC3-GFP⁺ puncta. H4-LC3-GFP cells were treated with rapamycin (0.2 µM) and MBCQ (5 µM) as indicated. The image data are expressed as % of control vehicle treated cells. 1000 cells were analyzed per treatment condition.

(C) H4-LC3-GFP cells were treated with rapamycin (0.2 µM) and MBCQ (10 µM) as indicated for 2 hr and the cell lysates were analyzed by western blotting using anti-LC3. β-tubulin was used as a control.

(D) An active (C43=spautin-1) and an inactive (C71) derivatives of MBCQ.

(E) MEF cells were treated with DMSO (1‰), rapamycin (0.2 µM) alone, or together with MBCQ (10 µM), C43 (10 µM) or C71 (10 µM) for 4 hr. The cell lysates were analyzed for western blotting using anti-LC3 antibody. β-tubulin was used as a loading control.

(F) Dose-response (in µM) of C43 and inactive C71. H4-LC3-GFP cells were treated with rapamycin (0.2 µM) for 12 hr with C43 or C71 as indicated. The LC3-GFP⁺ puncta were quantified as in (B). Autophagy index =%{[total LC3-GFP⁺ spot intensity (compound+rapamycin treated) per cell] – [total LC3-GFP⁺ spot intensity (DMSO treated) per cell]} / {[total LC3-GFP⁺ spot intensity (rapamycin treated) per cell] – [total LC3-GFP⁺ spot intensity (DMSO treated) per cell]}. Rap = rapamycin.

(G) H4-LC3-GFP cells were treated with spautin-1(10 µM) with or without E64D (5 µM) for indicated periods of time. The cell lysates were analyzed by western blotting using anti-LC3 and anti-β-tubulin.

All error bars indicate STD. See also Figure S1 and S2.