Silva for technical advice, and Andreas Villunger, Julio A

Silva for technical advice, and Andreas Villunger, Julio A. PIDDosome thus sequentially integrates DNA damage and mitotic checkpoint signals to decide cell Mouse monoclonal to XRCC5 fate in response to genotoxic stress. We further show that by sequestering PIDD at the kinetochore, BubR1 acts to delay PIDDosome formation until the next cycle, defining a new mechanism by which cells evade apoptosis during mitosis. Introduction The PIDDosome is a caspase-activation platform whose significance remains unclear more than a decade after its biochemical isolation by Tschopp and colleagues (Bock et al., 2012; Janssens and Tinel, 2012; Kumar, 2009; Tinel and Tschopp, 2004). Initial Prodigiosin views of the complex as a stress-inducible, proapoptotic device have been supported by studies implicating the PIDDosome in cell death responses to DNA damage and other stimuli (Ando et al., 2012; Berube et al., 2005; Jelinek et al., 2013; Niizuma et al., 2008). However, there are experimental settings in which one or more PIDDosome components show inconsistent phenotypes (Kim et al., 2009; Manzl et al., 2009; Manzl et al., 2012; Ribe et al., 2012). Further impeding the functional elucidation of the complex, the identities of the PIDDosomes upstream regulators and downstream substrates remain essentially unknown. The PIDDosome comprises the death domain (DD) proteins, PIDD (heterozygous MEF lines in which mutationally impaired BubR1 acetylation reduces total BubR1 levels to variable degrees (Park et al., 2013). Reduction of BubR1 was sufficient to trigger caspase-2 cleavage after IR, the extent of which correlated with the severity of BubR1 reduction (Figure 1E, compare lanes 4 and 6). To assess the PIDDosome-dependence of these effects, we depleted BubR1 from mutant zebrafish embryos, all apoptosis induced by IR+Chk1i depends on caspase-2 (Figure 2C, compare bars 2 and 17) (Sidi et al., 2008). Open in a separate window Figure 2 BubR1 suppresses PIDDosome-mediated apoptosis(A) HeLa cells transfected with the indicated siRNAs or Go6976 (1 M) were treated with or without IR (10 Gy) (black and grey bars respectively), harvested 24 hr post IR stained for TUNEL and analyzed by flow cytometry. Data are means +/? SD of 3 independent experiments. *p 0.05, ns, non-significant; two-tailed Students t-test. (B) HeLa cells were transfected with the indicated siRNAs, treated with or without 10 Gy IR (black and grey bars respectively), and stained with alamar Blue at 72 hr post IR. Data are means +/? SD of 3 independent experiments. **p 0.01, two-tailed Students t-test. (C) HeLa cells stably expressing the indicated shRNAs were transfected with the indicated siRNAs or Go6976 (1 M), treated with IR(10 Gy), harvested 24 hr post IR and stained for TUNEL. Data are means +/? SD of 3 independent experiments. Significance vs. corresponding shGFP controls: *p 0.05, **p 0.01, ns, non-significant; two-tailed Students t-test. (DCO) zebrafish embryos were non-injected or injected at the one-cell stage with standard control (MOs, incubated 17 hr later with or without G?6976 at indicated concentrations (M), treated with or without 15 Gy IR and stained with the cell death marker acridine orange (AO) after 7 hours. All embryos imaged live at 24hpf. (P) Quantification of AO stains shown in (DCO). White bars, uninjected; gray bars, MO; black bars, MO. Data collected from 3 independent experiments ( 10 embryos per condition). All data are reported as means SEM (two-tailed Students t-test). (Q) RT-PCR of and transcripts from embryos injected with MO or MO. Note the nonsense-mediated decay of transcript in the MO injected embryos. (R) HeLa cells transfected with the indicated siRNAs were treated with DMSO or Go6976 (0.5M) with or without IR (10 Gy) and harvested 24 hr post IR. Lysates were analyzed by western blot. See also Figure S2 Similar to Chk1i, siRNA depletions of BubR1, Bub1 and Aurora B triggered a robust, PIDDosome-dependent apoptotic response to IR in otherwise radioresistant HPV+ HeLa cells or SV-40 MEFs (Figures 2ACC and S2A). In contrast, knockdowns of Mad2 or Rad51, which have no effect on caspase-2 cleavage (Figures 1B and S1A), failed to trigger apoptosis after IR (Figure 2A). These results indicated that PIDDosome control by BubR1, Bub1 and Aurora B is biologically significant and, again, independent of their canonical MCC signaling function. We next tested the in vivo relevance of these observations in the zebrafish system, in which the caspase-2 apoptotic response to IR+Chk1i was originally identified (Sidi et al., 2008). As expected from this study, 18-hour post-fertilization (hpf) mutant embryos failed to respond to IR unless Chk1 was simultaneously inhibited (Figures 2E, G; quantification of all acridine orange stains is shown in Figure 2P). While morpholino (MO) knockdown of the zebrafish orthologue, MEFs in which BubR1 localization at KTs is.Confocal microscopy was performed using a Leica TCS SP5 II Confocal over an inverted microscope. Tschopp, 2004). Initial views of the complex as a stress-inducible, proapoptotic device have been supported by studies implicating the PIDDosome in cell death responses to DNA damage and other stimuli (Ando et al., 2012; Berube et al., 2005; Jelinek et al., 2013; Niizuma et al., 2008). However, there are experimental settings in which one or more PIDDosome components show inconsistent phenotypes (Kim et al., 2009; Manzl et al., 2009; Manzl et al., 2012; Ribe et al., 2012). Further impeding the functional elucidation of the complex, the identities of the PIDDosomes upstream regulators and downstream substrates remain essentially unknown. The PIDDosome comprises the death domain (DD) proteins, PIDD (heterozygous MEF lines in which mutationally impaired BubR1 acetylation reduces total BubR1 levels to variable degrees (Park et al., 2013). Reduction of BubR1 was sufficient to trigger caspase-2 cleavage after IR, the extent of which correlated with the severity of BubR1 reduction (Figure 1E, compare lanes 4 and 6). To assess the PIDDosome-dependence of these effects, we depleted BubR1 from mutant zebrafish embryos, all apoptosis induced by IR+Chk1i depends on caspase-2 (Figure 2C, compare bars 2 and 17) (Sidi et al., 2008). Open in a separate window Figure 2 BubR1 suppresses PIDDosome-mediated apoptosis(A) HeLa cells transfected with the indicated siRNAs or Go6976 (1 M) were treated with or without IR (10 Gy) (black and grey bars respectively), harvested 24 hr post IR stained for TUNEL and analyzed by circulation cytometry. Data are means +/? SD of 3 self-employed Prodigiosin experiments. *p 0.05, ns, non-significant; two-tailed College students t-test. (B) HeLa cells were transfected with the indicated siRNAs, treated with or without 10 Gy IR (black and grey bars respectively), and stained with alamar Blue at 72 hr post IR. Data are means +/? SD of 3 self-employed experiments. **p 0.01, two-tailed College students t-test. (C) HeLa cells stably expressing the indicated shRNAs were transfected with the indicated siRNAs or Proceed6976 (1 M), treated with IR(10 Gy), harvested 24 hr post IR and stained for TUNEL. Data are means +/? SD of 3 self-employed experiments. Significance vs. related shGFP settings: *p 0.05, **p 0.01, ns, non-significant; two-tailed College students t-test. (DCO) zebrafish embryos were non-injected or injected in the one-cell stage with standard control (MOs, incubated 17 hr later with or without G?6976 at indicated concentrations (M), treated with or without 15 Gy IR and stained with the cell death marker acridine orange (AO) after 7 Prodigiosin hours. All embryos imaged live at 24hpf. (P) Quantification of AO staining demonstrated in (DCO). White colored bars, uninjected; gray bars, MO; black bars, MO. Data collected from 3 self-employed experiments ( 10 embryos per condition). All data are reported as means SEM (two-tailed College students t-test). (Q) RT-PCR of and transcripts from embryos injected with MO or MO. Notice the nonsense-mediated decay of transcript in the MO injected embryos. (R) HeLa cells transfected with the indicated siRNAs were treated with DMSO or Proceed6976 (0.5M) with or without IR (10 Gy) and harvested 24 hr post IR. Lysates were analyzed by western blot. Observe also Number S2 Much like Chk1i, siRNA depletions of BubR1, Bub1 and Aurora B induced a powerful, PIDDosome-dependent apoptotic response to IR in normally radioresistant HPV+ HeLa cells or SV-40 MEFs (Numbers 2ACC and S2A). In contrast, knockdowns of Mad2 or Rad51, which have no effect on caspase-2 cleavage (Numbers 1B and S1A), failed to result in apoptosis after IR (Number.