Journal: Cellular and Molecular Life Sciences

Article Title: USP48 and A20 synergistically promote cell survival in Helicobacter pylori infection

doi: 10.1007/s00018-022-04489-7

Figure Lengend Snippet: USP48 controls A20 de novo synthesis and suppresses caspase cleavages. a AGS cells were transfected with siRNA against USP48 and infected with H. pylori for the indicated times. Total RNA was isolated at the indicated times and analysed using quantitative RT-PCR for the TNFAIP3 transcript (gene of A20). Data shown depict the average of triplicate determinations normalized to GAPDH housekeeping gene. Error bars denote mean ± SD. b , c AGS cells were transfected with siRNA against USP48 and infected with H. pylori for indicated times. Whole-cell lysates were subjected to IB for analysis of the indicated proteins. C-Casp8 or C-Casp3 = cleaved caspases. d AGS cells were transfected with recombinant human USP48 and infected with H. pylori for 24 h. Whole-cell lysates were subjected to IB analysis of the indicated proteins. e AGS cells were transfected with siRNA against USP48 and infected with H. pylori for the indicated times. IP with an anti-Caspase-8 antibody was performed at the indicated times in the presence of NEM and OPT, followed by IB analysis of the indicated proteins. f AGS cells transfected with siRNA were infected with H. pylori for 24 h before incubation with Caspase-Glo ® 8 reagent for 1 h. Luminescence of caspase-8 activity was measured and calculated as a fold increase compared to the uninfected scramble control. g AGS cells were transfected with siRNA against USP48 and infected with H. pylori for 24 h, followed by staining with annexin V/PI. Apoptotic cell death was analysed by flow cytometry. Data shown depict the average of two independent experiments. Error bars denote mean ± SD. h AGS cells were transfected with siRNA against USP48 and infected with H. pylori for 24 h. Cleaved caspase-3/7 expression was detected by the IncuCyte ® S3 Live-Cell Analysis Image system. Scale bars = 100 µm. Data shown depict the average of four pictures from distinct regions. Error bars denote mean ± SD. Data information: Data shown in ( a ) are from three independent experiments with three technical replicates. Data shown in ( b – e , g ) are representative for at least two independent experiments. Data shown in ( f ) are from one experiment with three technical replicates. Data shown in ( h ) are from one experiments with four technical replicates. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 (Student’s t -test)

Article Snippet: The following primary antibodies were used: A20 (sc-166692), C23 (sc-13057), CSN6 (sc-137153), Elongin B (sc-11447), Lamin B2 (sc-377379), RelA (sc-8008) and Ubquitin (sc-8017) were purchased from Santa Cruz Biotechnology; Caspase 3 (#9662), Caspase 8 (#9746), Cleaved Caspase 3 (#9661), IκBα (#4812), phospho-RelA (#3031) were purchased from Cell Signalling Technology; CSN2 (ab155774) and USP48 (ab72226) were purchased from Abcam; GAPDH (#MAB374) and Ubiquitin K63 linkage (05-1308)) were purchased from Millipore; CSN1 (BML-PW8285-0100, ENZO); CSN5 (GTX70207, GeneTex); FLAG (#F3165, Sigma-Aldrich).

Techniques: Transfection, Infection, Isolation, Quantitative RT-PCR, Recombinant, Incubation, Activity Assay, Control, Staining, Flow Cytometry, Expressing, Cell Analysis

Journal: Cellular and Molecular Life Sciences

Article Title: USP48 and A20 synergistically promote cell survival in Helicobacter pylori infection

doi: 10.1007/s00018-022-04489-7

Figure Lengend Snippet: USP48-suppressed caspase cleavage is A20-dependent. a AGS cells were transfected with siRNA against A20, and subsequently transfected with recombinant human USP48. One hour after transfection, the cells were infected with H. pylori for indicated times. Whole-cell lysates were subjected to IB for analysis of the indicated proteins. b AGS cells were transfected with siRNA against A20, and subsequently transfected with recombinant human USP48. One hour after transfection, the cells were infected with H. pylori for 24 h. Cleaved caspase-3/7 expression was detected by the IncuCyte ® S3 Live-Cell Analysis Image system. Scale bars = 100 µm. Data shown depict the average of four pictures from distinct regions. Error bars denote mean ± SD. c AGS cells were transfected with siRNA against USP48 and then transfected with recombinant human A20 protein. One hour after protein transfection, cells were infected with H. pylori for the indicated times. Total cell lysates were subjected to IB analysis of the indicated proteins. d Schematic representation of the findings in this study. Infection of H. pylori induces fast activation of NF-κB, leading to nuclear translocation of RelA (1) and expression of the target gene TNFAIP3 (encodes for A20) (2). Termination of RelA activity by ECS socs1 -dependent ubiquitinylation and degradation (3). CSN-associated USP48 deubiquitinylates RelA-Ub resulting in RelA stabilisation (4) and prolonged A20 de novo synthesis (5). USP48 and A20 synergistically suppresses caspase-8 activity and apoptotic cell death (6). Data information: Data shown in ( a , c ) are representative for at least two independent experiments. Data shown in ( b ) are from one experiments with four technical replicates. * P ≤ 0.05, *** P ≤ 0.001 (Student’s t -test)

Article Snippet: The following primary antibodies were used: A20 (sc-166692), C23 (sc-13057), CSN6 (sc-137153), Elongin B (sc-11447), Lamin B2 (sc-377379), RelA (sc-8008) and Ubquitin (sc-8017) were purchased from Santa Cruz Biotechnology; Caspase 3 (#9662), Caspase 8 (#9746), Cleaved Caspase 3 (#9661), IκBα (#4812), phospho-RelA (#3031) were purchased from Cell Signalling Technology; CSN2 (ab155774) and USP48 (ab72226) were purchased from Abcam; GAPDH (#MAB374) and Ubiquitin K63 linkage (05-1308)) were purchased from Millipore; CSN1 (BML-PW8285-0100, ENZO); CSN5 (GTX70207, GeneTex); FLAG (#F3165, Sigma-Aldrich).

Techniques: Transfection, Recombinant, Infection, Expressing, Cell Analysis, Activation Assay, Translocation Assay, Activity Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: A20 plays a critical role in the immunoregulatory function of mesenchymal stem cells

doi: 10.1111/jcmm.12849

Figure Lengend Snippet: Inflammatory cytokines induce A20 expression in MSC s. MSC s ( A ) and C3H/10T1/2 ( B and C ) were treated with 0, 2, 5 and 10 ng/ml IFN ‐γ and TNF ‐α for 24 hrs. A20 mRNA and protein levels were examined by qRT ‐ PCR and Western blot analysis. MSC s ( D ) and C3H/10T1/2 ( E and F ) were treated with 5 ng/ml IFN ‐γ and TNF ‐α for 0, 6, 12 and 24 hrs, and mRNA and protein expression levels were determined by qRT ‐ PCR and Western blot analysis, respectively.

Article Snippet: After 24 hrs, C3H/10T1/2 cells were transduced with lentivirus expressing murine A20 shRNA (shA20 C3 MSCs) or control lentivirus (shCTRL C3 MSCs) in the presence of 10 μg/ml polybrene (Santa Cruz Biotechnology, Dallas, TX, USA) for 6 hrs.

Techniques: Expressing, Quantitative RT-PCR, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: A20 plays a critical role in the immunoregulatory function of mesenchymal stem cells

doi: 10.1111/jcmm.12849

Figure Lengend Snippet: Morphological and phenotypic characterization of C3 MSC s after A20 knockdown. ( A ) qRT ‐ PCR analysis of A20 mRNA levels with or without A20 knockdown. ( B ) Morphology and ( C ) size of cultured sh CTRL C3 MSC s and shA20 C3 MSC s were analysed by microscopy and flow cytometry. ( D ) Cell surface markers were analysed by flow cytometry, ** P < 0.01. All experiments were repeated three times.

Article Snippet: After 24 hrs, C3H/10T1/2 cells were transduced with lentivirus expressing murine A20 shRNA (shA20 C3 MSCs) or control lentivirus (shCTRL C3 MSCs) in the presence of 10 μg/ml polybrene (Santa Cruz Biotechnology, Dallas, TX, USA) for 6 hrs.

Techniques: Knockdown, Quantitative RT-PCR, Cell Culture, Microscopy, Flow Cytometry

Journal: Journal of Cellular and Molecular Medicine

Article Title: A20 plays a critical role in the immunoregulatory function of mesenchymal stem cells

doi: 10.1111/jcmm.12849

Figure Lengend Snippet: A20 knockdown attenuates immunosuppressive capacity of C3 MSC s in vitro . CFSE ‐labelled CD 3 + T cell was cultured alone ( A ) or co‐cultured with different numbers of C3 MSC s ( B ) in RPMI 1640 complete medium supplemented with PMA (50 ng/ml) and ionomycin (1 μg/ml) for 48 hrs. Cells were subjected to flow cytometry for T cell proliferation as detected by the CFSE signal.

Article Snippet: After 24 hrs, C3H/10T1/2 cells were transduced with lentivirus expressing murine A20 shRNA (shA20 C3 MSCs) or control lentivirus (shCTRL C3 MSCs) in the presence of 10 μg/ml polybrene (Santa Cruz Biotechnology, Dallas, TX, USA) for 6 hrs.

Techniques: Knockdown, In Vitro, Cell Culture, Flow Cytometry

Journal: Journal of Cellular and Molecular Medicine

Article Title: A20 plays a critical role in the immunoregulatory function of mesenchymal stem cells

doi: 10.1111/jcmm.12849

Figure Lengend Snippet: A20 knockdown inhibits tumorigenesis in vivo . ( A ) Representative tumours photographed 13 days after C57 BL /6 mice were injected with PBS , sh CTRL C3 MSC s or shA20 C3 MSC s. ( B ) On day 0, 5 × 10 5 B16‐F0 cells in 100‐μl PBS were injected subcutaneously into the right posterior flanks, with or without co‐injection of sh CTRL C3 MSC s or shA20 C3 MSC s (1 × 10 6 cells). Tumour growth was measured at 2‐day intervals, and tumour volume was calculated. ( C ) Thirteen days later, all mice were killed and tumours were sectioned and weighted. Each treatment group included four mice, and data are representative of three independent experiments, ** P < 0.01.

Article Snippet: After 24 hrs, C3H/10T1/2 cells were transduced with lentivirus expressing murine A20 shRNA (shA20 C3 MSCs) or control lentivirus (shCTRL C3 MSCs) in the presence of 10 μg/ml polybrene (Santa Cruz Biotechnology, Dallas, TX, USA) for 6 hrs.

Techniques: Knockdown, In Vivo, Injection

Journal: Journal of Cellular and Molecular Medicine

Article Title: A20 plays a critical role in the immunoregulatory function of mesenchymal stem cells

doi: 10.1111/jcmm.12849

Figure Lengend Snippet: A20 knockdown showed no significant effect on ICAM ‐1, VCAM ‐1, PD ‐L1, PGE 2 or nitric oxide expression. Sh CTRL C3 MSC s and shA20 C3 MSC s were cultured with or without stimulation with 5 ng/ml IFN ‐γ and TNF ‐α. 12 hrs later, the expression of ( A ) ICAM ‐1, ( B ) VCAM ‐1 and ( C ) PD ‐L1 were analysed by flow cytometry. ( D ) Nitric oxide production was measured by Griess assay. ( E ) PGE 2 production was measured by ELISA .

Article Snippet: After 24 hrs, C3H/10T1/2 cells were transduced with lentivirus expressing murine A20 shRNA (shA20 C3 MSCs) or control lentivirus (shCTRL C3 MSCs) in the presence of 10 μg/ml polybrene (Santa Cruz Biotechnology, Dallas, TX, USA) for 6 hrs.

Techniques: Knockdown, Expressing, Cell Culture, Flow Cytometry, Griess Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Cellular and Molecular Medicine

Article Title: A20 plays a critical role in the immunoregulatory function of mesenchymal stem cells

doi: 10.1111/jcmm.12849

Figure Lengend Snippet: A20 inhibits TNF ‐α and promotes IL ‐10 production in C3 MSC s, and the p38/ MAPK pathway is involved in A20‐induced immunomodulation. TNF ‐α ( A ) and IL ‐10 ( B , left) expression was examined with or without stimulation with 5 ng/ml IFN ‐γ and TNF ‐α by qRT ‐ PCR . IL ‐10 protein level was determined by ELISA ( B , right). ( C ) The time courses of p38 phosphorylation in response to 5 ng/ml IFN ‐γ and TNF ‐α was determined by immunoblotting, ** P < 0.01, * P < 0.05.

Article Snippet: After 24 hrs, C3H/10T1/2 cells were transduced with lentivirus expressing murine A20 shRNA (shA20 C3 MSCs) or control lentivirus (shCTRL C3 MSCs) in the presence of 10 μg/ml polybrene (Santa Cruz Biotechnology, Dallas, TX, USA) for 6 hrs.

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Phospho-proteomics, Western Blot