1. Volume 46, Issue 2, Pages 200-211 (April 2012)
Inflammasome-Activated Caspase 7 Cleaves PARP1 to Enhance the Expression of a Subset of NF-κB Target Genes 
Süheda Erener, Virginie Pétrilli, Ingrid Kassner, Roberta Minotti, Rosa Castillo, Raffaella Santoro, Paul O. Hassa, Jürg Tschopp, Michael O. Hottiger 
Molecular Cell 
Volume 46, Issue 2, Pages (April 2012)
DOI: /j.molcel
Copyright © 2012 Elsevier Inc. Terms and Conditions

2. Figure 1
PARP1 Cleavage at D214 Regulates Expression of a Subset of NF-κB Target Genes
(A) LPS-stimulated induction of CSF2, IL-6, LIF, and IP-10 expression in peritoneal macrophages isolated from WT and D214N PARP1 mice. Cells were stimulated with LPS for 1 hr and mRNA levels were determined by real-time RT-PCR analysis. Samples were normalized to Rps12 expression levels and expressed as fold increase relative to unstimulated mRNA levels. Data are means ± SEM of at least four independent representative experiments.
(B) Induction of IL-6 and IP-10 expression in stably complemented THP1 cells. Cells were stimulated with LPS for 1 hr and mRNA levels were determined by real-time RT-PCR analysis. Samples were normalized to Rpl28 expression levels and expressed as fold increase relative to unstimulated mRNA levels. Data are means ± SD, n = 2.
(C and D) LPS stimulation does not affect cell viability. THP1 macrophages were stimulated with LPS for the indicated times and (C) stained with calcein and ethidium homodimer and analyzed by immunofluorescence microscopy (n = 3–7, mean ± SEM) or (D) assayed for LDH release as indicated in the experimental procedures (n = 3, mean ± SEM).
(E) THP1 cells were stimulated with camptothethcin (CPT; 10 μM for 6 hr), total cell extracts were prepared and proteins were analyzed by western blot. Ctrl∗ is the sample treated with etoposide for 16 hr.
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2 Characterization of WT and D214N PARP1
(A) ChIP analysis of p65 recruitment upon LPS stimulation on the target promoters. WT (white bars) and D214N (gray bars) PARP1 peritoneal macrophages were stimulated with LPS for the indicated times. p65 was immunoprecipitated, and the occupancy on the indicated promoters was measured by qRT-PCR. Data are represented as bound/input and normalized to values corresponding to unstimulated conditions. Unstimulated levels were arbitrarily set as 1. Prolactin promoter was assessed as negative control. Data represent means from 3–4 independent experiments ± SEM.
(B) Enzymatic activity of recombinant PARP1 proteins. WT, D214N, and E988K PARP1 baculovirus generated proteins were incubated with or without DNA for 5 min, and ribosylation was analyzed by autoradiography.
(C) Acetylation of WT and D214N PARP1 (AcCoA=Acetyl coenzyme A).
(D) PARP1 interaction with p65 after LPS stimulation in WT and D214N peritoneal macrophages. Cells were stimulated with LPS and then fractionated. Nuclear extracts were subjected to western blotting.
(E) Interaction of PARP1 fragments with GST-p65. GST-p65 recombinant protein purified from insect cells was incubated with full-length WT and D214N PARP1 proteins. Interaction was analyzed by western blot.
(F) Bone marrow-derived macrophages from wild-type mice were stimulated with LPS for different times in the presence or absence of Z-VAD-fmk (50 μM). Total cell extracts were prepared, and PARP1 cleavage was analyzed by western blot.
(G) Gene expression profiles of CSF2, IL-6, and IP-10 in Raw cells. Cells were treated with LPS (1 μg/ml) and/or Z-VAD-fmk (10 μM) for 4 hr and mRNA levels were determined by real-time RT-PCR analysis. Samples were normalized to Rps12 expression levels and expressed as fold increase relative to unstimulated mRNA levels. Data are means ± SEM of three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

4. Figure 3
Caspase 7 Cleaves PARP1 at D214 In Vitro and Controls NF-κB Gene Induction
(A) Screening of caspase family members (1–10) for PARP1 cleavage at D214 in vitro. Recombinant PARP1 WT or D214N mutant recombinant proteins were incubated with active caspases (1–10) for 20 min at 30°C in a caspase cleavage buffer. Reaction products were analyzed by SDS gel followed by western blot using PARP1 antibody.
(B) THP1 cells were stimulated with LPS for the indicated times. Cells were then fractionated, and cytoplasmic and nuclear extracts were subjected to western blotting.
(C) CSF2, LIF, and IP10 fold induction upon LPS stimulation of caspase 3 and 7 knockdown macrophages. Cells were stimulated with LPS for 1 hr and gene expression was analyzed by real-time RT-PCR. Samples were normalized to Rps12 and expressed as fold increase relative to unstimulated mRNA levels. Data are means ± SD, n = 2.
(D) IL-6 protein levels released by IP macrophages were determined by ELISA after 5 hr of LPS induction (IL-6 could not be detected in uninduced peritoneal macrophages). Values represent means ± SD of two measurements.
(E) IL-6 expression is significantly reduced in siCasp7-treated THP1 cells. Changes in gene expression upon LPS stimulation were expressed as fold response. Fold response was set arbitrarily as 100% in simock transfected cells. Data are means ± SD, n = 2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 4 Caspase 7 Is a Target of the NLRP3 Inflammasome
(A) PARP1 cleavage upon LPS stimulation is mediated by caspase 1 and caspase 7. Peritoneal macrophages from wild-type, caspase 1 −/−, and caspase 7 −/− mice were stimulated for the indicated times with LPS, total cell extracts were prepared, and PARP1 cleavage and caspase 1 and caspase 7 activation were analyzed by western blot.
(B) In IP macrophages lacking the essential inflammasome adaptor ASC, IL-6 protein levels upon LPS stimulation were strongly reduced as determined by ELISA (means ± SEM).
(C) BMDM cells from wild-type and NLRP3 −/− mice were stimulated for the indicated times with LPS, total cell extracts were prepared, and PARP1 cleavage and caspase 1 and caspase 7 activation were analyzed by western blot.
(D) Peritoneal macrophages from wild-type mice were stimulated for the indicated times with LPS or MSU. Total cell extracts (XT) or the culture media (SN) were analyzed by western blot.
(E) Uncleavable PARP1 (D214N) does not interfere with caspase activation by LPS. Peritoneal macrophages from wild-type and D214N PARP1 mice were stimulated for the indicated time with LPS, total cell extracts were prepared, and PARP1 cleavage and caspase 1 and caspase 7 activation were analyzed by western blot.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions

6. Figure 5 PARP1 Cleavage Regulates the Expression of IL-6 in THP1 Cells
(A) Kinetics of PARP1 occupancy upon LPS stimulation on the target promoters by ChIP analysis. Raw cells were stimulated with LPS for different times. PARP1 was immunoprecipitated with a PARP1 antibody, and the occupancy on the indicated promoters was measured by real-time RT-PCR. Results are normalized to values corresponding to the beads control (0 hr time point), and the means ± SEM are shown.
(B) Effect of Z-VAD-fmk on PARP1 occupancy on the target promoters after LPS stimulation. Raw cells were treated for 2 hr with LPS and Z-VAD-fmk. The same analysis was performed as in (A). Results are from two independent experiments, ± SD.
(C) Kinetics of caspase 7 occupancy at the IL-6 promoter. Values are represented as bound/input and were normalized to the IgG control. The mean ± SD of two RT-PCR quantifications is shown, and two independent experiments were performed.
(D) ChIP analysis of PARP1 occupancy upon LPS stimulation on the target promoters in WT and D214N PARP1-complemented THP1 cells. Cells were stimulated with LPS for 1 hr. PARP1 was immunoprecipitated with a myc antibody, and the occupancy on the indicated promoters was measured by real-time RT-PCR. Results are corrected for the input controls and expressed as relative occupancy, unstimulated levels being set arbitrarily as 1. H2B promoter was assessed as negative control. Data are means ± SD, n = 2.
(E) Chromatin accessibility was assessed by real-time PCR (CHART-PCR). Nuclei from unstimulated and LPS-stimulated BMDMs were incubated with MNase. (−1) kb upstream promoter and TSS of CSF2 were analyzed. Results are expressed as uncut over cut genomic DNA and normalized to unstimulated levels. Unstimulated uncut/cut genomic DNA level around TSS was arbitrarily set as 100. Data are from a pool of six mice, and means ± SD are shown (n = 2).
(F) After stimulation of BMDMs with LPS for 1 hr, mRNA levels were determined by real-time RT-PCR analysis. Samples were normalized to Rps12 expression levels and expressed as fold increase relative to unstimulated mRNA levels. Data are means ± SEM, three independent mice groups where each group contains two mice, n = 6.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2012 Elsevier Inc. Terms and Conditions