Journal: Apoptosis

Article Title: Identification and validation of uterine stimulant methylergometrine as a potential inhibitor of caspase-1 activation

doi: 10.1007/s10495-017-1405-z

Figure Lengend Snippet: Identification of the modulators of the ASC-mediated activation of PC1. a Scheme of the 96-well plate assays performed to identify inhibitors. Primary screening: ASC-dependent PC1 activation was monitored in vitro by the release of AFC from fluorogenic PC1 substrate Ac-WEHD-AFC in the presence of 880 FDA-approved compounds (20 µM) from the Prestwick Chemical Library. Secondary screening: caspase-1 activation was monitored in vitro by the release of AFC from fluorogenic PC1 substrate Ac-WEHD-AFC in the presence of the positive hits (20 µM) obtained in the previous assay. b Graphical representation of the behavior of the three selected compounds in the primary ( black bars ) and secondary screening ( white bars ) at 20 µM in DMSO. A Student’s t -test statistical analysis (***p < 0.01) was performed to compare the caspase-1 inhibition for each compound in the presence or absence of ASC. Data represent the mean ± SD of three experiments

Article Snippet: Samples were separated in a 14% SDS-PAGE gel, transferred to a nitrocellulose membrane and blocked with 5% skimmed milk for 1 h. Then the membrane was incubated overnight with primary antibodies: α-casp1 (1:1000; Cell Signaling #2225) and α-IL-1β (1:1000; Acris #R1130P) at 4 °C.

Techniques: Activation Assay, In Vitro, Inhibition

Journal: Apoptosis

Article Title: Identification and validation of uterine stimulant methylergometrine as a potential inhibitor of caspase-1 activation

doi: 10.1007/s10495-017-1405-z

Figure Lengend Snippet: MEM inhibits the NLRP1-dependent inflammasome in THP-1 cells. IL-1β ( a ) and IL-18 ( b ) secretion was evaluated by the ELISA technique upon activation of the NLRP3 inflammasome with LPS (100 ng/ml) and MDP (50 µg/ml). Cells were treated with MEM at 10, 50 or 100 µM, and with VX-765 (VX) at 5 µM. c THP-1 cells were stimulated as described above, and supernatants and inputs were analyzed by Western blotting; IL-1β and cleaved caspase-1 (C1) were detected. The shown data are representative of three independent experiments. d Measurement of the release of LDH into the extracellular medium under the above-described conditions. C−: cells non-treated and C+: cells treated with stimuli activation but without the anti-inflammatory treatment. Asterisks represent significant differences to the positive control (C+), as determined by a one-way ANOVA test with Dunnetts’s multiple post test comparison **p < 0.05. All the data are expressed as the mean ± SD of three experiments

Article Snippet: Samples were separated in a 14% SDS-PAGE gel, transferred to a nitrocellulose membrane and blocked with 5% skimmed milk for 1 h. Then the membrane was incubated overnight with primary antibodies: α-casp1 (1:1000; Cell Signaling #2225) and α-IL-1β (1:1000; Acris #R1130P) at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot, Positive Control, Comparison

Journal: Apoptosis

Article Title: Identification and validation of uterine stimulant methylergometrine as a potential inhibitor of caspase-1 activation

doi: 10.1007/s10495-017-1405-z

Figure Lengend Snippet: MEM inhibits the NLRP3-dependent inflammasome in THP-1 cells. IL-1β ( a ) and IL-18 ( b ) secretion was evaluated by the ELISA technique upon activation of the NLRP3 inflammasome with LPS (100 ng/ml) and ATP (2.5 mM). Cells were treated with MEM at 10, 50 or 100 µM, and with VX-765 (VX) at 5 µM. c THP-1 cells were stimulated as described above, and supernatants and inputs were analyzed by Western blotting; IL-1β and cleaved caspase-1 (C1) were detected. The shown data are representative of three independent experiments. d Measurement of the release of LDH into the extracellular medium under the above-described conditions. C−: cells non-treated and C+: cells treated with stimuli activation but without the anti-inflammatory treatment. Asterisks represent significant differences to the positive control (C+), as determined by a one-way ANOVA test with Dunnetts’s multiple post test comparison **p < 0.05. All the data are expressed as the mean ± SD of three experiments

Article Snippet: Samples were separated in a 14% SDS-PAGE gel, transferred to a nitrocellulose membrane and blocked with 5% skimmed milk for 1 h. Then the membrane was incubated overnight with primary antibodies: α-casp1 (1:1000; Cell Signaling #2225) and α-IL-1β (1:1000; Acris #R1130P) at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot, Positive Control, Comparison