Fig 2.

G9a inhibition results in formation of autophagosomes but not autophagic flux. (A) Panc04.03 cells were treated over time with 3 μM YT-2-6, 5 μM UNC0638, or 250 nM torin for 12 h, and lysates were immunoblotted with the indicated antibodies. (B) Immunoblot analysis for LC3B lipidation in ATG5^−/− mouse fibroblasts treated with diluent or 3 μM YT-2-6 for 12 h. (C) Pancreatic SU86.86 cells were analyzed by immunofluorescence for LAMP1 (red) along with p62 (green) after treatment with 3 μM YT-2-6 for 12 h. The nucleus was visualized by Hoechst staining (blue). (D) Immunoblot analysis of SU86.86 cells treated for 12 h with a 3 μM concentration of the G9a inhibitor YT-2-6, the TORC inhibitors torin (250 nM) and rapamycin (100 nM), or combinations as indicated shows that although p62 is upregulated upon G9a inhibition, it does not get degraded within autolysosomes unless G9a inhibition is combined with TORC inhibition. (E) Analysis of dually tagged GFP-mCherry-p62 to study autophagic flux in HeLa cells upon treatment with 3 μM YT-2-6 and/or 250 nM torin for 12 h. (F) Colocalization coefficients were determined from the results in panel E for mCherry overlap with GFP (lower colocalization indicates mCherry fluorescence independent of GFP and exposure of the tagged p62 to an acidic compartment).