Fig. 2

Proteasome inhibitor-mediated stabilization of NOXA is abrogated through an autophagy-driven proteolysis. a Increased half-life of Noxa protein after bortezomib treatment is diminished after palbociclib co-treatment. MCL cell line Mino was treated with 100 nM palbociclib for 16 h and subsequently co-treated with 8 nM bortezomib. After 8 h co-treatment, 20 μg/ml cycloheximide was added to the cells and samples were harvested 0, 15, 30, 45, 60, and 90 min after cycloheximide exposition for Western blot analysis. b Palbociclib treatment induces autophagy. MCL cell line Mino was treated with 100 nM palbociclib for 16 h and subsequently co-treated with 8 nM bortezomib. After 8 h, protein expression was analyzed (upper panel). MCL cell line Mino was treated with 40 μM hydroxychloroquine and 100 nM palbociclib for 16 h and subsequently co-treated with 8 nM bortezomib. After 8 h, protein expression was analyzed (middle panel). MCL cell line Mino was treated with 40 μM hydroxychloroquine for 24 h or treated with 100 nM palbociclib for 16 h and subsequently co-treated with 8 nM bortezomib for 24 h. After treatment, autophagic vesicles were measured by Cyto-ID staining (lower panel). c Knockdown of autophagy-related genes reverses the palbociclib-mediated antagonism on bortezomib-mediated stabilization of NOXA. MCL cell line Mino was transfected with siRNA targeting ATG5 and ATG7. Twenty-four hours after transfection, cells were treated with 100 nM palbociclib for 16 h and subsequently co-treated with 8 nM bortezomib. After 8 h, cells were exposed to 20 μg/ml cycloheximide and samples were harvested 0, 15, 30, 45, 60, and 90 min after cycloheximide addition and analyzed by Western blot. d Knockdown of autophagy-related genes as well as autophagy inhibitors counteract palbociclib-mediated antagonism on bortezomib-induced cell death and NOXA induction. MCL cell line Mino was transfected with siRNA targeting ATG5 and ATG7. Twenty-four hours after transfection, cells were treated with 100 nM palbociclib for 16 h and subsequently co-treated with 8 nM bortezomib. After 8 h, protein expression was analyzed (upper right) and cell death was assessed by AnnexinV-PI staining 24 h post-treatment (upper left). MCL cell line Mino was treated with 100 nM palbociclib and 20 μM liensinine (upper middle panel), 5 μM Spautin-1 (lower middle panel), or 2 mM 3-MA (lower panel) for 16 h and subsequently co-treated with 8 nM bortezomib. After 8 h, protein expression was analyzed (right) and cell death was assessed by AnnexinV-PI staining 24 h post-treatment (left). Data represent means ± S.D. from three independent experiments