1. Volume 9, Issue 3, Pages 910-917 (November 2014)
Granzyme A Produces Bioactive IL-1β through a Nonapoptotic Inflammasome- Independent Pathway 
Dagmar Hildebrand, Konrad A. Bode, David Rieß, Daniela Cerny, Anna Waldhuber, Franziska Römmler, Julia Strack, Simone Korten, Joachim H.C. Orth, Thomas Miethke, Klaus Heeg, Katharina F. Kubatzky 
Cell Reports 
Volume 9, Issue 3, Pages (November 2014)
DOI: /j.celrep
Copyright © 2014 The Authors Terms and Conditions

2. Cell Reports 2014 9, 910-917DOI: (10.1016/j.celrep.2014.10.003)
Copyright © 2014 The Authors Terms and Conditions

3. Figure 1 PMT Induces Transcription and Processing of Pro-IL-1β
RAW264.7 cells were stimulated with 6.5 nM PMTwt and PMT C1165S for 18 hr or with LPS (100 ng/ml) for 3 hr.
(A) Quantitative RT-PCR using primers for Il1b was performed (mean ± SD; n = 3).
(B) Cells were stimulated with PMTwt and PMT mutants (18 hr) or with LPS/ATP (100 ng/ml, 4 hr; 5 mM, 2 hr) and western blots were performed (n = 3).
(C) Released IL-1β was measured with ELISAs. The indicated SD was calculated from triplicates of one experiment, n = 3.
Additional controls of the experimental setup can be found in Figure S1.
Cell Reports 2014 9, DOI: ( /j.celrep )
Copyright © 2014 The Authors Terms and Conditions

4. Figure 2 PMT Induces NF-кB Activation TLR Independently through RhoA
(A) RAW264.7 cells were stimulated with PMT or LPS, cytoplasmic and nuclear lysates were prepared, and p65 was detected. As control, β-actin and B23 were detected. Experiments were repeated at least two times.
(B) EMSA analysis. DNA-protein complexes were separated by electrophoresis and detected using a p65 antibody. Experiments were repeated at least two times.
(C–F) BMCs from Myd88-deficient, Trif-deficient, and and wild-type (WT) mice were isolated and macrophages were generated. After stimulation with PMT (18 hr) or LPS (4 hr), RT-PCRs were performed (C and D). Supernatants of cells treated with PMT (18 hr) or LPS/ATP (4 hr/2 hr) were used in IL-1β-ELISAs (E and F).
(G) Cells were transfected with an NF-кB-reporter construct, treated with Ly27632 (15 μM) either in parallel with PMT or 3 hr prior to LPS, and stimulated overnight. Transcriptional activity was analyzed, and results are presented as induction of the unstimulated sample (C–G mean ± SD; n = 3).
(H) Cells were treated as described, and IL-1β was determined in western blots (n = 3).
Cell Reports 2014 9, DOI: ( /j.celrep )
Copyright © 2014 The Authors Terms and Conditions

5. Figure 3
PMT-Triggered Processing of Pro-IL-1β Is Mediated by GrzA and Occurs Independently of Caspase-1
(A) RAW264.7 cells were stimulated with PMT or LPS/ATP for the indicated time. Cleavage of caspase-1 was detected by western blot analysis (n = 5).
(B) BMDMs were generated from Casp1-deficient and WT mice, stimulated with PMT (18 hr) or LPS/ATP (4 hr/2 hr), and used for western blot analysis (n = 3).
(C) Supernatant from (B) was used in IL-1β-ELISAs.
(D) Mice were injected intraperitoneally with PMT (10 ng/g, 6.5 hr), LPS (25 μg/g, 3 hr), or PBS (mock). One-third of the isolated liver was incubated in medium at 37°C for 24 hr. The amount of IL-1β and caspase-1 was detected by western blot analysis in the medium supernatant (n = 3).
(E) RAW264.7 cells or BMDMs were stimulated overnight with PMT or LPS. GrzA-ELISAs were performed.
(F) RAW264.7 cells were treated with 20 nM of JAK-inhibitor I 2 hr prior to PMT (6 hr) or LPS (4 hr) stimulation. Gzma sequence was measured by quantitative RT-PCR.
(G) Lysates of supernatants and cells that were treated with 20 nM of JAK-inhibitor I 2 hr prior to PMT (overnight) or LPS/ATP (4 hr/2 hr) were produced and western blot analyses were performed (n = 3).
(H and I) BMDMs from Gmza- and Gmzb-deficient and WT mice were stimulated with PMT (o.n.) or LPS/ATP (4 hr/2 hr). In (H), lysates were analyzed by immunoblotting (n = 3).
(I) Supernatant was used in IL-1β-ELISAs.
(J) EL4.NOB-1 cells were cultured with supernatants of PMT- or LPS/ATP-stimulated RAW264.7 cells or stimulated with PMT, LPS/ATP, or recombinant IL-1β. IL-2 was measured with ELISAs.
In (C), (E), (F), (I), and (J), data are mean ± SD (n = 3). Figure S2 shows further experiments regarding caspase-1, GrzA, and other bacterial toxins.
Cell Reports 2014 9, DOI: ( /j.celrep )
Copyright © 2014 The Authors Terms and Conditions

6. Figure 4
GrzA Enters Target Cells Perforin Independently and Does Not Induce Cytotoxicity
(A and B) RAW264.7 cells were treated with 30 μM brefeldin (BFA) 2 hr before stimulation with PMT (overnight), only with PMT or BFA, or with LPS/ATP (4 hr/2 hr) ± BFA. IL-1β was detected by immunoblotting (A) (n = 3). Supernatants were used for GrzA-ELISAs (B).
(C) THP-1 cells were differentiated with 500 ng/ml PMA into macrophages, stimulated with LPS (2 hr), and treated with 100 ng/ml human recombinant GrzA overnight. Controls: unstimulated, LPS, huGrzA, PMT. IL-1β was detected by ELISAs (B and C, mean ± SD; n = 3).
(D) Human blood-derived macrophages were generated and treated with PMT, recombinant GrzA (100 ng/ml), staurosporine (1 mM), doxorubicin (1 μM), or LPS/ATP. Cells were stained with Annexin V-FITC and propidium iodide (PI) and analyzed by flow cytometry. A caspase-3/7 assay is shown in Figure S3.
Cell Reports 2014 9, DOI: ( /j.celrep )
Copyright © 2014 The Authors Terms and Conditions