Regulatory Mechanisms for sOb-R Generation Figure 4. Caspase-mediated apoptosis, induced by incubation with staurosporine, activates Ob-R shedding mainly mediated by ADAM10. (A) sOb-R levels in the supernatant of Ob-Rfl or Ob-R219.3 transfected cells after incubation with staurosporine (0.1–0.5 mM) or DMSO (0.1%) for 24 h and 48 h. sOb-R levels of staurosporine-treated cells are displayed relative to sOb-R levels of cells treated with DMSO (B) sOb-R levels in the supernatant of Ob-R219.3 transfected cells after pre-incubation with the broad spectrum caspase inhibitor Z-VAD(OMe)FMK (100 mM) or DMSO (0.1%) for 30 min and subsequent stimulation with staurosporine (0.1–0.5 mM) or DMSO (0.1%) for 24 h and 48 h. sOb-R levels of staurosporine- or Z- VAD(OMe)FMK-treated cells are shown relative to those of cells treated with DMSO. (C) Representative western blot of cleaved caspase-3 and cleaved PARP after staurosporine incubation. Ob-R219.3 transfected cells were pre-incubated with Z-VAD(OMe)FMK (100 mM) or DMSO (0.1%) for 30 min and subsequently stimulated with staurosporine (0.1–0.5 mM) or DMSO (0.1%) for 48 h. Following this incubation cleaved caspase-3 and cleaved PARP in cell lysates was determined by western blot analysis. PC, positive control (cell lysate of Jurkat cells treated with cytochrome c). (D) sOb-R levels in the supernatant of HEK cells transfected with a non-targeting, ADAM10 or ADAM17 specific siRNA (50 nM) and subsequently with Ob-R219.3 after incubation with staurosporine (0.5 mM) or DMSO (0.1%) for 48 h. sOb-R levels of ADAM10 and ADAM17 siRNA transfected cells are presented relative to sOb-R levels of cells transfected with a non-targeting siRNA and treated with DMSO. Data are presented as means 6 SD of n$3 experiments. doi:10.1371/journal.pone.0034787.g004 Ob-R219.3 or Ob-Rfl and GFP (vector control, equimolar to Ob- R219.3) co-transfected cells were serum starved for 16 h and afterwards stimulated with leptin (100 ng/ml) for 30 min. Fig. 6A illustrates that the co-expression of Ob-Rfl and Ob-R219.3 reduced leptin-induced STAT3 activation by 62.2% (P,0.01)compared to cells transfected with Ob-Rfl + GFP only. To clarify the influence of increasing sOb-R levels on leptin signaling we pre-incubated medium containing 10 ng/ml leptin with sOb-R (1–1000 ng/ml) for one hour at 37uC. Afterwards the medium was applied to stimulate 16 h serum-starved HEK cells for 30 min. Fig. 6 B shows that increasing sOb-R concentrations reduced leptin-mediated STAT3 phosphorylation compared to Ob-Rfl transfected cells incubated with medium free of sOb-R. An effect of 20–94% inhibition was detectable for the addition of 100– 1000 ng/ml sOb-R, whereas total STAT3 levels remained unchanged. PLoS ONE | www.plosone.org 7 April 2012 | Volume 7 | Issue 4 | e34787