We thank Hermann Steller for thoughtful conversation around the manuscript and Gal Sevi Karniel for excellent graphical work

We thank Hermann Steller for thoughtful conversation around the manuscript and Gal Sevi Karniel for excellent graphical work. an E3-ligase for Bcl-2 and ARTS is essential for this process. Collectively, these results suggest a distinct model for the regulation of Bcl-2 by ARTS-mediated degradation. ubiquitylation of Bcl-2 occurs upon induction of apoptosis. Both MEFs and HeLa cells pretreated with MG132 showed accumulation of poly-ubiquitylated Bcl-2 upon induction of apoptosis (Physique 1D). The appearance of poly-ubiquitylated Bcl-2 was correlated with decreased Bcl-2 levels in apoptotic cells (Physique 1D). This suggests that Bcl-2 is usually down-regulated through UPS-mediated degradation during apoptosis. Open in a separate window Physique 1 Bcl-2 protein levels are down-regulated by the ubiquitin-proteasome system during apoptosisA. Apoptosis was induced Curculigoside in HeLa and immortalized MEFs using STS for the indicated occasions, and endogenous Bcl-2 was detected by Western blot analysis. B. Apoptosis was induced Curculigoside in HeLa, BT-549 and COS-7 cells with etoposide. NT (No Treatment). In main MEFs apoptosis was induced with 100 M etoposide for 5 h. A decrease in endogenous Bcl-2 levels was seen upon treatment with both STS and etoposide. C. Apoptosis was induced in HeLa cells using STS in the presence or absence of 20 M of MG132. Western and densitometry analyses revealed decreased levels of Bcl-2 with STS treatment, and stabilization of Bcl-2 upon MG132 treatment. This suggests that Bcl-2 levels are down-regulated via the UPS. D. WT MEFs and HeLa cells were transiently transfected with Bcl-2, XIAP and ubiquitin and treated with 20 M MG132 for 6 h and with 1.75 M STS. IP with anti-Bcl-2 was followed by Western Blotting with anti-ubiquitin antibodies. *Represents the IG heavy chain. Poly-ubiquitylated forms of Bcl-2 appeared in apoptotic cells and correlated with decreased Bcl-2 levels. Open in a separate window Physique 2 ARTS is required for down-regulation of Bcl-2 levels in the cytosolAI. HeLa ARTS knockdown (ARTS KD) cells and Sept4/ARTS KO MEFs show significantly higher levels of steady-state Bcl-2 protein when compared with WT cells. This suggests that ARTS plays an important role in regulating Bcl-2 levels. B. WT and ARTS KD HeLa cells were treated with 1.75 M STS. Western Blot analyses demonstrate that while decreased Bcl-2 levels were seen in apoptotic WT HeLa cells, Bcl-2 levels in ARTS KD HeLa cells remained unchanged. C. Western Blot analyses of cytosolic fractions of BT-549, HeLa WT and HeLa ARTS KD cells uncover that endogenous Bcl-2 is found in the cytosol of WT STS-treated cells. In contrast, a strong inhibition in translocation of Bcl-2 to the cytosol was seen in ARTS KD HeLa cells. This suggests that ARTS is required for the proper translocation of Bcl-2 from mitochondria to the cytosol upon apoptotic induction. D. Immuno-fluorescence (IF) was performed on HeLa and a stable Bcl-2 knockdown (Bcl-2 KD) cells. The portion of cells with cytosolic staining of ARTS is usually Curculigoside represented in the bar chart. While only a small portion of WT untreated (NT) HeLa cells show the presence of ARTS in the cytosol, a significant increase in cells made up of cytosolic ARTS was seen following STS treatment. In contrast, the majority of HeLa Bcl-2 KD NT cells exhibit cytosolic ARTS (four fold higher than WT HeLa cells), and, only a slight increase in cells with cytosolic ARTS is seen after STS treatment. (* * p-value 0.01). Observe also supplemental Physique S1. E. Cytosolic and mitochondrial fractions of WT MEFs and Bcl-2 KO MEFs were analyzed by WB Curculigoside analysis with COX IV as a mitochondrial and GAPDH as a cytosolic marker. In Bcl-2 KO MEFs, the majority of ARTS was in the cytosol. This suggests that Bcl-2 is usually involved in localizing ARTS to mitochondria. F. IF of WT MEFs and Sept4/ARTS KO MEFs transiently transfected with GFP-Bcl-2. Cellular localization of Bcl-2 was quantified and the portion of cells with cytosolic Bcl-2 is usually shown in the bar charts. While a significant increase in cytosolic Bcl-2 was seen in apoptotic WT MEFs, Curculigoside the levels of cytosolic Bcl-2 in Sept4/ARTS KO MEFs remained unchanged. Observe also supplemental Physique S1. G. Subcellular fractionation of HeLa cells was followed by in vivo ubiquitylation of each portion. IgG represents the control cells incubated with non-specific IgG. Poly-ubiquitylated forms of Bcl-2 were seen only in the cytosolic portion. * Represents the IG heavy chain. This indicates that ubiquitylation of Sema3d Bcl-2 occurs in the cytosol and that ARTS is required for translocation of Bcl-2 to the cytosol during apoptosis. ARTS is required for down-regulation of Bcl-2 levels in the cytosol High levels of ARTS are sufficient to promote apoptosis in a variety of cell lines, and inactivation of ARTS protects against apoptosis.