caspase 1 knockdown kd  (InvivoGen)

Journal: eLife

Article Title: T3SS translocon induces pyroptosis by direct interaction with NLRC4/NAIP inflammasome

doi: 10.7554/eLife.100820

Figure Lengend Snippet:

Article Snippet: Cell line ( H. sapiens ) , THP1-Casp1-KD , InvivoGen , thp-dcasp1 , Casp1 knockdown.

Techniques: Control, Knockdown, Knock-Out, Transfection, Construct, shRNA, Gene Knockout, Recombinant, Negative Control, Plasmid Preparation, Sequencing, Cytotoxicity Assay, Enzyme-linked Immunosorbent Assay, Software

Journal: Israel journal of chemistry

Article Title: Pharmacologic Targeting of PDIA1 Inhibits NLRP3 Inflammasome Assembly and Activation

doi: 10.1002/ijch.202300125

Figure Lengend Snippet: A . Representative fluorescent microscopy images of ASC-GFP cells untreated, treated with LPS (1 μg/mL) for 16 h ± nigericin (1 μM) 3 hours and in the presence or absence of AA147 (10 μM). ASC-GFP specks are indicated with white arrows. B . Quantification of images shown in (A). ****p<0.0001 using a Brown-Forsythe and Welch ANOVA test with Dunnett-T3 correction for multiple comparisons. Error bars show SD. C . Quantification of secreted bioactive IL-1β assessed using the SEAP Quanti-blue assay. HEK Blue IL-1β cells were treated with media conditioned on THP1 monocytes pre-treated with AA147 (10 μM) or vehicle for 16 h and stimulated with LPS (1 μg/mL) and nigericin (1 μM) for 24 h relative to an unstimulated, vehicle treated control. ****p<0.0001, ***p<0.001, **p<0.01 for one-way ANOVA test with Dunnett correction for multiple comparisons. Error bars show SEM for n=3. D . ELISA quantifications of total IL-1β detected in media conditioned on THP1 monocyte-derived macrophages pre-treated with AA147 (10 μM) or vehicle for 16 h then stimulated with LPS (1 μg/mL) for 3 h and incubated with ATP (5 mM) for 24 h. ***p<0.001, **p<0.01 for one-way ANOVA test with Dunnett correction for multiple comparisons. Error bars show SEM for n=3.

Article Snippet: Initially, we monitored secretion of IL-1β from monocyte THP1 cells using HEK-Blue IL-1β reporter cells (Invivogen) – a cell line that expresses the IL-1β receptor and an NFκB-responsive secreted alkaline phosphatase (SEAP) reporter.

Techniques: Microscopy, Control, Enzyme-linked Immunosorbent Assay, Derivative Assay, Incubation

Journal: Israel journal of chemistry

Article Title: Pharmacologic Targeting of PDIA1 Inhibits NLRP3 Inflammasome Assembly and Activation

doi: 10.1002/ijch.202300125

Figure Lengend Snippet: A . Quantification of secreted bioactive IL-1β assessed using the SEAP Quanti-blue assay. HEK Blue IL-1β cells were treated with media conditioned on THP1 monocyte-derived macrophages pre-treated with AA147 (10 μM) or vehicle for 16 h in the presence or absence of the ATF6 inhibitor Ceapin-A7 (CP7; 6 μM) and subsequently stimulated for 3 h with LPS (1 μg/mL) and 24 h with ATP (5 mM). Results are relative to unstimulated, vehicle treated controls. ***p<0.001, ****p<0.0001 with multiple unpaired two-tailed t-tests. Error bars show SEM for n=3. B . Quantification of secreted bioactive IL-1β assessed using the SEAP Quanti-blue assay. HEK Blue IL-1β cells were treated with media conditioned on THP1 monocyte-derived macrophages pre-treated with AA147 (10 μM) or vehicle for 16 h in the presence or absence of the ATF6 inhibitor PF429242 (10 μM) and subsequently stimulated for 3 h with LPS (1 μg/mL) and 24 h with ATP (5 mM). Results are relative to unstimulated, vehicle treated controls. ***p<0.001, ****p<0.0001 with multiple unpaired two-tailed t-tests. Error bars show SEM for n=3. C . mRNA expression measured using qPCR, of the pro-inflammatory cytokine IL1B in THP1 monocyte-derived macrophages treated for 16 h with vehicle or AA147 (10 μM) and subsequently stimulated with LPS (1 μg/mL) for 3 h. Error bars show 95 % CI. D . Immunoblot of inflammasome components in THP1 monocyte-derived macrophages pre-treated with vehicle or AA147 (10 μM) for 16 h and subsequently stimulated with vehicle or LPS (1 μg/mL) for 3 h.

Article Snippet: Initially, we monitored secretion of IL-1β from monocyte THP1 cells using HEK-Blue IL-1β reporter cells (Invivogen) – a cell line that expresses the IL-1β receptor and an NFκB-responsive secreted alkaline phosphatase (SEAP) reporter.

Techniques: Derivative Assay, Two Tailed Test, Expressing, Western Blot

Journal: Israel journal of chemistry

Article Title: Pharmacologic Targeting of PDIA1 Inhibits NLRP3 Inflammasome Assembly and Activation

doi: 10.1002/ijch.202300125

Figure Lengend Snippet: A . Quantification of secreted bioactive IL-1β assessed using the SEAP Quanti-blue assay. HEK Blue IL-1β cells were treated with media conditioned on THP1 monocytes stably expressing shRNAs of the respective target PDI. were stimulated for 3 h with LPS (1 μg/mL) and 24 h with ATP (5 mM) or nigericin (1 μM). *p<0.05, ***p<0.001, ****p<0.0001 two-way ANOVA test with Dunnett correction for multiple comparisons. Error bars show SD. B . Relative number of ASC-GFP specks per cell detected using fluorescent microscopy in undifferentiated THP1 ASC-GFP monocyte cells stably expressing either a scramble shRNA or a PDIA1 shRNA (PDIA1 KD ). Cells were stimulated with LPS (1 μg/mL) for 16 h and nigericin (5 μM) for 3 h. Quantification is relative to the overall average number of specks / total cells across all wells in LPS (1 μg/mL) and nigericin (5 μM) stimulated wells. ****p<0.0001 using a Brown-Forsythe and Welch ANOVA test with Dunnett-T3 correction for multiple comparisons. Error bars show SD. C . Caspase-1 activity assessed using Caspase-1 Glo assay from PDIA1 KD or scramble THP1 monocytes stimulated for 16 h with LPS (1 μg/mL) and 45 min nigericin (1 μM). Error bars show SD; each dot represents an individual replicate relative to LPS. ****p<0.0001 using a two-way ANOVA. D . Immunoblot of cell lysates from PDIA1 KD or scramble THP1 cells stimulated with LPS (1 μg/mL) for 3 h and nigericin (5 μM) for 1 h. ZVAD-VAD-FMK (50 μM) was used as a positive control. E . Quantification of protein levels measured using immunoblots from cell lysates from PDIA1 KD or scramble THP1 cells stimulated with LPS (1 μg/mL) for 3 h. *p<0.05, ***p<0.001. ****p<0.0001 using multiple unpaired two-tailed t-tests. Error bars show SEM for n=3.

Article Snippet: Initially, we monitored secretion of IL-1β from monocyte THP1 cells using HEK-Blue IL-1β reporter cells (Invivogen) – a cell line that expresses the IL-1β receptor and an NFκB-responsive secreted alkaline phosphatase (SEAP) reporter.

Techniques: Stable Transfection, Expressing, Microscopy, shRNA, Activity Assay, Glo Assay, Western Blot, Positive Control, Two Tailed Test

Journal: Israel journal of chemistry

Article Title: Pharmacologic Targeting of PDIA1 Inhibits NLRP3 Inflammasome Assembly and Activation

doi: 10.1002/ijch.202300125

Figure Lengend Snippet: A . Chemical structures of the PDI inhibitors KSC-34 and RB-11-CA. B . Relative number of ASC-GFP specks per cell detected using fluorescent microscopy in undifferentiated THP1 ASC-GFP expressing monocyte cells pre-treated with KSC-34 (20 μM) and stimulated with LPS (1 μg/mL) for 16 h followed by a treatment of nigericin (5 μM) for 3 h. Quantification is relative to the overall average number of specks / total cells across all wells in LPS (1 μg/mL) and nigericin (5 μM) stimulated wells. ****p<0.0001 using a Brown-Forsythe and Welch ANOVA test with Dunnett-T3 correction for multiple comparisons. Error bars show SD. B . Rhodamine fluorescence and Coomassie staining of an SDS-PAGE gel of lysates from THP1 cells treated for 16 h with AA147 alk (10 μM), KSC-34 (20 μM), or RB-11-CA (20 μM). C . Relative number of ASC-GFP specks per cell detected using fluorescent microscopy in undifferentiated THP1 ASC-GFP expressing monocyte cells pre-treated with KSC-34 (20 μM) for 16 h then stimulated with LPS (1 μg/mL) for 3 h and nigericin for 3 h (5 μM). ****p<0.0001 using a Brown-Forsythe and Welch ANOVA test with Dunnett-T3 correction for multiple comparisons. Error bars show SD. D . Quantification of secreted bioactive IL-1β assessed using the SEAP Quanti-blue assay. HEK Blue IL-1β cells were treated with media conditioned on THP1 monocytes pre-treated with KSC-34 (20 μM) or RB-11-CA (20 μM) and subsequently stimulated for 3 h with LPS (1 μg/mL) and 24 h with nigericin (1 μM) relative to an unstimulated, vehicle treated control. *p< 0.05, ***p<0.001, ****p<0.0001 with one-way ANOVA test with Dunnett correction for multiple comparisons. Error bars show SEM for n=3.

Article Snippet: Initially, we monitored secretion of IL-1β from monocyte THP1 cells using HEK-Blue IL-1β reporter cells (Invivogen) – a cell line that expresses the IL-1β receptor and an NFκB-responsive secreted alkaline phosphatase (SEAP) reporter.

Techniques: Microscopy, Expressing, Fluorescence, Staining, SDS Page, Control