Journal: Molecular cell

Article Title: Liquid Phase Separation of NEMO Induced by Polyubiquitin Chains Activates NF-κB

doi: 10.1016/j.molcel.2022.03.037

Figure Lengend Snippet:

Article Snippet: Rabbit monoclonal anti-A20 , Cell Signaling Technology , Cat# 5630.

Techniques: Western Blot, Immunostaining, Recombinant, Software

Journal: Cell Death & Disease

Article Title: miR-21 promotes NLRP3 inflammasome activation to mediate pyroptosis and endotoxic shock

doi: 10.1038/s41419-019-1713-z

Figure Lengend Snippet: a – e A20 ( a ), Nlrp3 ( b ), Asc ( c ), Casp1 ( d ), and IL-1b ( e ) mRNA levels in LPS-treated WT and miR-21 KO BMDMs. f A20, IL-1b and NLRP3 inflammasome protein levels in LPS-treated WT and miR-21 KO BMDMs after 4 h. g Sequence alignment of the A20 3′-UTR with miR-21 across multiple species. h Activity of luciferase reporters containing WT or mutant A20 3′-UTRs that were used with miR-21 mimics or respective controls to cotransfect HEK-293T cells. i BMDMs were treated with 500 ng/ml LPS for different lengths of time. Cell extracts were immunoblotted for IKBα and p-IKBα. j , k WT and miR-21 KO BMDMs were left unstimulated (control) or stimulated with LPS (500 ng/ml), ATP (5 mM), or nigericin (20 mM) alone or stimulated with LPS (500 ng/ml) for 4 h and then treated with 5 mM ATP or 20 mM nigericin for 30 min. Culture supernatants were analyzed for IL-6 secretion ( j ) and TNF secretion ( k ). The data represent the mean ± SD of one among three biological replicates, with three technical replicates each (* P < 0.05; ** P < 0.01; Student’s t test)

Article Snippet: Immunoblots were incubated overnight with primary antibodies against NLRP3 (AG-20B-0014-C100, Adipogen, CH), ASC (AG-25B-0006, Adipogen, CH), mouse caspase-1 (AG-20B-0042-C100, Adipogen, CH), human caspase-1 (AG-20B-0048-C100, Adipogen, CH), IkBa (4814, Cell Signaling, USA), phospho-IkBa (2859, Ser32) (Cell Signaling, USA), A20 (23456-1-AP, Proteintech, USA), IL-1β (12242, Cell Signaling, USA) human GSDMD (sc-81868, Santa Cruz Biotechnology, USA), mouse GSDMD (ab209845, Abcam, USA), and GAPDH (10494-1-AP, Proteintech, USA).

Techniques: Sequencing, Activity Assay, Luciferase, Mutagenesis, Control

Journal: Cell Death & Disease

Article Title: miR-21 promotes NLRP3 inflammasome activation to mediate pyroptosis and endotoxic shock

doi: 10.1038/s41419-019-1713-z

Figure Lengend Snippet: a – d A20 ( a ), Nlrp3 ( b ), Asc ( c ), and Casp1 ( d ) mRNA levels in LPS-treated miR-21 KO BMDMs transfected with control or A20 siRNA. e Protein levels of NLRP3 inflammasomes in LPS-treated miR-21 KO BMDMs transfected with control or A20 siRNA. f Activation of caspase-1 in LPS-primed and ATP- or nigericin-treated miR-21 KO BMDMs transfected with control or A20 siRNA. g Supernatants were analyzed for IL-1β in ( f ). h BMDMs were treated with 500 ng/ml LPS for different lengths of time in miR-21 KO BMDMs transfected with control or A20 siRNA. Cell extracts were immunoblotted for IKBα and p-IKBα. The data represent the mean ± SD of one among three biological replicates, with three technical replicates each (* P < 0.05; ** P < 0.01; Student’s t test)

Article Snippet: Immunoblots were incubated overnight with primary antibodies against NLRP3 (AG-20B-0014-C100, Adipogen, CH), ASC (AG-25B-0006, Adipogen, CH), mouse caspase-1 (AG-20B-0042-C100, Adipogen, CH), human caspase-1 (AG-20B-0048-C100, Adipogen, CH), IkBa (4814, Cell Signaling, USA), phospho-IkBa (2859, Ser32) (Cell Signaling, USA), A20 (23456-1-AP, Proteintech, USA), IL-1β (12242, Cell Signaling, USA) human GSDMD (sc-81868, Santa Cruz Biotechnology, USA), mouse GSDMD (ab209845, Abcam, USA), and GAPDH (10494-1-AP, Proteintech, USA).

Techniques: Transfection, Control, Activation Assay

Journal: Cell Death & Disease

Article Title: miR-21 promotes NLRP3 inflammasome activation to mediate pyroptosis and endotoxic shock

doi: 10.1038/s41419-019-1713-z

Figure Lengend Snippet: a Imaging assay of pyroptosis in WT and miR-21 KO BMDMs treated as indicated. Scale bars: 100 μm. b Immunoblotting demonstrates cleavage of GSDMD. WT and miR-21 KO BMDMs stably expressing Flag–GSDMD were stimulated as indicated. c Immunoblotting with mouse antibody for GSDMD demonstrates cleavage of GSDMD in WT and miR-21 KO BMDMs treated with ATP or nigericin. d Immunoblotting demonstrates cleavage of GSDMD in WT and miR-21 KO BMDMs treated with A20 siRNA. GSDMD-FL full-length GSDMD, GSDMD-N N-terminal cleavage products of GSDMD. All data are representative of three independent experiments

Article Snippet: Immunoblots were incubated overnight with primary antibodies against NLRP3 (AG-20B-0014-C100, Adipogen, CH), ASC (AG-25B-0006, Adipogen, CH), mouse caspase-1 (AG-20B-0042-C100, Adipogen, CH), human caspase-1 (AG-20B-0048-C100, Adipogen, CH), IkBa (4814, Cell Signaling, USA), phospho-IkBa (2859, Ser32) (Cell Signaling, USA), A20 (23456-1-AP, Proteintech, USA), IL-1β (12242, Cell Signaling, USA) human GSDMD (sc-81868, Santa Cruz Biotechnology, USA), mouse GSDMD (ab209845, Abcam, USA), and GAPDH (10494-1-AP, Proteintech, USA).

Techniques: Imaging, Western Blot, Stable Transfection, Expressing

Journal: Cell Death & Disease

Article Title: miR-21 promotes NLRP3 inflammasome activation to mediate pyroptosis and endotoxic shock

doi: 10.1038/s41419-019-1713-z

Figure Lengend Snippet: a , b Analysis of one published human septic shock clinical data set (GSE26440) and quantification of miR-21 expression from corresponding datasets. ** P < 0.01. The statistical significance of the differences was estimated using nonparametric two-sided Mann–Whitney tests. c PMA-differentiated THP-1 cells were transfected with control siRNA and a miR-21 inhibitor followed by stimulation with 500 ng/ml LPS for 4 h. Cell extracts were immunoblotted for A20 and NLRP3 inflammasomes. d , e PMA-differentiated THP-1 cells were transfected with control siRNA and a miR-21 inhibitor followed by stimulation with 500 ng/ml LPS for 4 h and treatment with ATP for 30 min. Cell extracts were immunoblotted for caspase-1 activation and GSDMD cleavage. f Human PBMCs were transfected with control siRNA and a miR-21 inhibitor followed by stimulation with 500 ng/ml LPS for 4 h. Cell extracts were immunoblotted for A20 and NLRP3 inflammasomes. g , h Human PBMCs were transfected with control siRNA and a miR-21 inhibitor followed by stimulation with 500 ng/ml LPS for 4 h and treatment with ATP for 30 min. Cell extracts were immunoblotted for caspase-1 activation and GSDMD cleavage

Article Snippet: Immunoblots were incubated overnight with primary antibodies against NLRP3 (AG-20B-0014-C100, Adipogen, CH), ASC (AG-25B-0006, Adipogen, CH), mouse caspase-1 (AG-20B-0042-C100, Adipogen, CH), human caspase-1 (AG-20B-0048-C100, Adipogen, CH), IkBa (4814, Cell Signaling, USA), phospho-IkBa (2859, Ser32) (Cell Signaling, USA), A20 (23456-1-AP, Proteintech, USA), IL-1β (12242, Cell Signaling, USA) human GSDMD (sc-81868, Santa Cruz Biotechnology, USA), mouse GSDMD (ab209845, Abcam, USA), and GAPDH (10494-1-AP, Proteintech, USA).

Techniques: Expressing, MANN-WHITNEY, Transfection, Control, Activation Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: STAT3 regulates inflammatory cytokine production downstream of TNFR1 by inducing expression of TNFAIP3 / A20

doi: 10.1111/jcmm.17489

Figure Lengend Snippet: The negative regulator of TNFR1 signalling, TNFAIP3/A20, is a rapidly induced STAT3 target gene. (A) Volcano plot showing differentially expressed genes in matched WT and STAT3 KO MEFs stimulated with TNF for 30 min measured by RNA‐sequencing. (B) Western blot of WT MEFs stimulated for 4 h with TNFR1aab in the presence of the STAT3 inhibitor Stattic (STAT3 inhibitor) or vehicle control (DMSO). (C) Quantification of data from three independent biological replicates of Panel B is shown in the bar graph and normalized to GAPDH.

Article Snippet: Transfer plasmid containing the A20 insert was obtained from Origene (MR210582L4, Tnfaip3 NM_009397, OriGene Technologies, Inc., MD, USA).

Techniques: RNA Sequencing, Western Blot, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: STAT3 regulates inflammatory cytokine production downstream of TNFR1 by inducing expression of TNFAIP3 / A20

doi: 10.1111/jcmm.17489

Figure Lengend Snippet: Effect of A20 expression on cytokine expression STAT3 KO MEFs. Cell culture media were collected from WT, STAT3 KO , or STAT KO MEFs transduced with a A20 expression plasmid and then stimulated with the TNFR1aab antibody for 4 h. Cytokine levels in the media were assayed using a multiplex cytokine array. (A) Cytokines whose levels were greater in media from STAT3 KO MEFS (i.e., suppressed by STAT3). (B) GM‐CSF whose level was reduced in media from STAT3 KO MEFs (i.e., induced by STAT3).

Article Snippet: Transfer plasmid containing the A20 insert was obtained from Origene (MR210582L4, Tnfaip3 NM_009397, OriGene Technologies, Inc., MD, USA).

Techniques: Expressing, Cell Culture, Transduction, Plasmid Preparation, Multiplex Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: STAT3 regulates inflammatory cytokine production downstream of TNFR1 by inducing expression of TNFAIP3 / A20

doi: 10.1111/jcmm.17489

Figure Lengend Snippet: Model of the mechanism whereby STAT3 limits chemokine expression downstream of TNFR1. (A) TNFR1 signalling in STAT3 KO MEFs. Signalling through TNFR1 activates Jak2. In the absence of STAT3, A20/Tnfaip3 is not induced; consequently, chemokine induction by NF‐κB is elevated relative to that in WT MEFs. (B) TNFR1 signalling in WT MEFs. In WT MEFs Jak2 activates STAT3, which translocates into the nucleus and binds the promoters of target genes. In conjunction with NF‐κB, STAT3 induces A20 expression. A20 acts as a negative feedback regulator of NF‐κB, inhibiting its activity. Consequently, the expression of chemokine targets of NF‐κB is diminished in WT MEFs relative to STAT3 KO MEFs. The figure was created with Biorender.com .

Article Snippet: Transfer plasmid containing the A20 insert was obtained from Origene (MR210582L4, Tnfaip3 NM_009397, OriGene Technologies, Inc., MD, USA).

Techniques: Expressing, Activity Assay

Journal: Leukemia

Article Title: Tumor suppressor TNFAIP3 (A20) is frequently deleted in Sézary syndrome.

doi: 10.1038/leu.2011.101

Figure Lengend Snippet: Figure 1 Fine tiling comparative genomic hybridization (FT-CGH) of chromosome 6q23-27. (a) FT-CGH results in six Se´zary syndrome patients and in the Se´zary syndrome-derived cell line SeAx showing TNFAIP3 (A20) deletions. The cutaneous T-cell lymphoma-derived cell line HuT-78 represents a non-deleted control. Three patients and the SeAx cell line showed bi-allelic deletions with the indicated minimal overlapping region of 90 kb. (b) Genome map (MapViewer; NCBI, http://www.ncbi.nlm.nih.gov/mapview/) of the minimal common region of biallelic deletions including a single gene: TNFAIP3 (13 818 8581–138 204 449 bp).

Article Snippet: A20 was amplified from pCR-TOPO-Blunt II-TOPO A20 (OriGene, Rockville, MD) using the primers TNFAIP3-fo (50-CT GGGACCATGGCACAACTC-30), TNFAIP3-re (50-CGGAAGGTT CCATGGGATTC-30) and cloned into the retroviral vector pMIGR.

Techniques: Hybridization, Derivative Assay, Control

Journal: Leukemia

Article Title: Tumor suppressor TNFAIP3 (A20) is frequently deleted in Sézary syndrome.

doi: 10.1038/leu.2011.101

Figure Lengend Snippet: Figure 2 A20 mRNA and protein expression in T-cell lines and Se´zary samples. (a) A20 mRNA and protein levels in T-cell leukemia cell line Jurkat and cutaneous T-cell lymphoma-derived cell lines HuT-78, MyLa and SeAx. The mRNA expression represents mean values (±s.d.) measured in four independent experiments. In western blot, Actin showed as a loading control. (b) A20 mRNA expression in sorted SS samples (mean of four technical replicates). Clinical samples: 245, 252, 313, 314 ( þ / þ ; no deletion), 247, 312 ( þ /; mono-allelic deletion), 308 (/; bi-allelic deletion). (c) A20 protein expression in sorted SS samples. Clinical samples: 313 (no deletion), 247, 312 (mono-allelic deletion), 308 (bi-allelic deletion); / þ stimulation mimicking T-cell receptor engagement (PMA, Ionomycin). GAPDH showed as a loading control.

Article Snippet: A20 was amplified from pCR-TOPO-Blunt II-TOPO A20 (OriGene, Rockville, MD) using the primers TNFAIP3-fo (50-CT GGGACCATGGCACAACTC-30), TNFAIP3-re (50-CGGAAGGTT CCATGGGATTC-30) and cloned into the retroviral vector pMIGR.

Techniques: Expressing, Derivative Assay, Western Blot, Control

Journal: Leukemia

Article Title: Tumor suppressor TNFAIP3 (A20) is frequently deleted in Sézary syndrome.

doi: 10.1038/leu.2011.101

Figure Lengend Snippet: Figure 3 A20 reconstitution in SeAx. The Se´zary cell line SeAx was transduced with a retroviral vector encoding A20 (MIGR-A20) or the empty vector MIGR as a negative control. (a) Transduction efficiency of mock-transduced SeAx (MIGR) and SeAx re-constituted for A20 were determined with EGFP measurement. (b) A20 protein expression in non-transduced SeAx (nt) and SeAx at 85 h and at 110 h after transduction. Actin represents a loading control. (c) Cell vitality of mock-transduced SeAx (MIGR) and SeAx re-constituted for A20 were determined using Annexin-V-APC/-AAD (mean from five independent experiments). (d) Proliferation activity of mock-transduced SeAx (MIGR) and SeAx re-constituted for A20 were determined with the BrdU incorporation assay. Mean percentage (±s.d.) of BrdU-positive cells was calculated from results obtained in five independent experiments.

Article Snippet: A20 was amplified from pCR-TOPO-Blunt II-TOPO A20 (OriGene, Rockville, MD) using the primers TNFAIP3-fo (50-CT GGGACCATGGCACAACTC-30), TNFAIP3-re (50-CGGAAGGTT CCATGGGATTC-30) and cloned into the retroviral vector pMIGR.

Techniques: Transduction, Retroviral, Plasmid Preparation, Negative Control, Expressing, Control, Activity Assay, BrdU Incorporation Assay

Journal: Transplant international : official journal of the European Society for Organ Transplantation

Article Title: Late outgrowth endothelial progenitor cells engineered for improved survival and maintenance of function in transplant-related injury.

doi: 10.1111/j.1432-2277.2011.01387.x

Figure Lengend Snippet: Figure 4 Effect of tumour necrosis factor (TNF)-a on late outgrowth endothelial progenitor cells (LO-EPC) activation. (a) Representative FACS plots of surface adhesion molecule (ICAM-1) expression in LO-EPC and human umbilical vein endothelial cells (HUVEC) in response to TNF-a stimu- lation. The empty histogram with dotted line represents untreated cells staining with isotype control antibody, the filled histogram represents untreated cells and the empty histogram with solid line represents cells treated with TNF-a (10 ng/ml) for 4 h. (b) TNF-a upregulates expression of inflammatory cytokines and chemokines in both LO-EPC and HUVEC. Relative expression levels in culture supernatants from LO-EPC and HUVEC following TNF-a stimulation for 4 h were measured using an R&D Cytokine Antibody Array kit and compared with supernatants from unstimu- lated cells. The data were analysed using Image J software and expressed as densitometric units (data represent mean ± SE of three experiments). (c) Phospho IjBa was used as indictor for NFjB activation and measured by Western blot of TNF-a treated cells. Time course of phospho IjBa expression measured by band intensity analysis of Western blots demonstrated a similar response to TNF-a stimulation in LO-EPC and HUVEC; b-actin protein expression is shown as a control. (d) Time course of endogenous A20 upregulation in response to TNF-a stimulation of LO-EPC as detected by Western blotting. (e) Time course of surface ICAM-1 expression in response to TNF-a stimulation of LO-EPC, as detected by flow cytometry. The filled histogram represents untreated cells and the empty histogram represent LO-EPC treated with TNF-a for the indicated time.

Article Snippet: Specific proteins were identified using monoclonal antibodies to human A20 (R&D Systems), human phosphorylated IjBa (Cell Signaling Technology, Danvers, MA, USA), and b-actin (Abcam Ltd, Cambridge, UK) followed by HRP-labelled secondary antibodies.

Techniques: Activation Assay, Expressing, Staining, Control, Ab Array, Software, Western Blot, Cytometry

Journal: Transplant international : official journal of the European Society for Organ Transplantation

Article Title: Late outgrowth endothelial progenitor cells engineered for improved survival and maintenance of function in transplant-related injury.

doi: 10.1111/j.1432-2277.2011.01387.x

Figure Lengend Snippet: Figure 7 Effect of overexpression of A20 on tumour necrosis factor (TNF)-a-induced apoptosis, angiogenesis and migration. (a) Overexpression of A20 significantly inhibited TNF-a-induced apoptosis in late outgrowth endothelial progenitor cells (LO-EPC) stimulated with TNF-a in the presence of cycloheximide (CHX), as determined by caspase 3 activity (measured using a luminescent substrate and expressed as relative light units). **P < 0.01 compared with LO-EPC transduced with empty vector (data represent mean ± SE of three independent experiments). (b) Overexpres- sion of A20 in LO-EPC did not affect their networks formation. Scale bar 200 lm. (c, d) LO-EPC overexpressing A20 and treated with TNF-a/CHX were able to maintain network formation (c), whereas LO-EPC transduced with empty vector showed disrupted network units under the same conditions (d). Scale bar 200 lm. (e) LO-EPC treated with TNF-a/CHX significantly impaired their ability to migrate. ##P < 0.01 compared with untreated LO-EPC. Overexpression of A20 in LO-EPC treated with TNF-a/CHX was able to restore their ability to migrate. **P < 0.01 compared with LO-EPC transduced with empty vector. Migration was determined by counting cells in the detection zone. The value was normalized against untreated cells. Data represent mean ± SE from three independent experiments.

Article Snippet: Specific proteins were identified using monoclonal antibodies to human A20 (R&D Systems), human phosphorylated IjBa (Cell Signaling Technology, Danvers, MA, USA), and b-actin (Abcam Ltd, Cambridge, UK) followed by HRP-labelled secondary antibodies.

Techniques: Over Expression, Migration, Activity Assay, Transduction, Plasmid Preparation