Regulatory Mechanisms for sOb-R Generation Figure 2. Constitutive shedding of human Ob-R isoforms is mainly mediated by ADAM10. (A) sOb-R levels in the supernatant of Ob- R219.3 transfected HEK cells after incubation with the metalloprotease inhibitors GI/GW (3 mM) or DMSO (0.1%) for 24 h. sOb-R levels of GI/GW incubated cells are presented relative to those of cells incubated with DMSO. (B and C) ADAM10 and ADAM17 mRNA (B) and protein expression (C) in lysates of HEK cells transfected with a specific siRNA for ADAM10 or ADAM17 or a non-targeting siRNA (50 nM). (D and E) sOb-R levels in the supernatant of HEK cells transfected with a specific siRNA for ADAM10, ADAM17 or a non-targeting siRNA (50 nM) and subsequently with Ob-Rfl (D) or Ob-R219.3 (E) followed by a further incubation under normal culture conditions for 24–72 h. sOb-R levels of ADAM10/ADAM17 siRNA transfected cells are shown relative to those of cells transfected with a non-targeting siRNA. Data are presented as means 6 SD of n$3 experiments. doi:10.1371/journal.pone.0034787.g002 transfected cells with staurosporine (0.1–0.5 mM) for 24 h and 48 h. This resulted in up to 2.31-fold (P,0.01) increased sOb-R levels compared to the non-treated control (Fig. 4 A) after 48 h. When investigating the long isoform Ob-Rfl the level of sOb-R remained comparable to control data (Fig. 4 A). Western blots for cleaved caspase-3 and cleaved Poly-(ADP-ribose) Polymerase (PARP) (Fig. 4C) and ELISA measurements of nucleosome release into the cytoplasm (Supporting information Fig. S4) proved successful induction of apoptosis by 0.5 mM staurosporine after 48 h. Pre-incubation with broad spectrum caspase inhibitor Z-VAD(OMe)-FMK(100 mM) impaired induction of apoptosis and diminished staurosporine-mediated PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e34787