Coatomer proteins modulate autophagy.

<p>(A) MDA-MB-231, MDA-MB-468, and SKOV3 cells stably overexpressing GFP-LC3 were transfected with siRNA targeting independent members of COPI for 72 h, and autophagosome formation was analyzed using the IN Cell analyzer 1000 system. Imatinib was used as a positive control for autophagosome formation. Results are the mean ± SD from triplicates. Representative graph of two independent experiments is shown. (B,C) Representative epifluorescent microscopy images (IN Cell Analyzer 1000) of GFP-LC3–positive vesicles in control MDA-MB-231 cells or after COPB2 depletion. (D) Immunoblot analysis of LC3 in MDA-MB-231 cells 72 h after siRNA mediated depletion of independent members of COPI. β-actin was used as a loading control. (E) MDA-MB-468 and U2OS cells treated with RF or COPB2 siRNA were assayed for protein levels of LC3. (F) MDA-MB-231 cells were transfected with siRNA against COPB2, COPG2 or risc-free (RF) and grown for the indicated times, lysed, and immunoblotted with antibodies to LC3, COPB2, COPG2 and β-actin. *, aspecific band.