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 Clinical Investigation

Article Title: MicroRNA-132 enhances transition from inflammation to proliferation during wound healing

doi: 10.1172/jci79052

Figure Lengend Snippet: Figure 8. Decreased proliferation and increased chemokine production of epidermal keratinocytes from miR-132–KO mice. (A) H&E staining of the skin from miR-132–KO mice (n = 9) and their WT littermate controls (n = 5) and epidermis thickness were analyzed. Scale bars: 100 μm. (B) Immunostaining of Ki-67 and HB-EGF in skin from WT (n = 9) and KO mice (n = 5). Sections were counterstained using DAPI (blue, nucleus). The number of Ki-67–positive cells was counted. Scale bars: 50 μm. (C) Epidermal keratinocytes were isolated from the skin of WT (n = 3) and KO (n = 3) mice. Ccl20, Cxcl5, Cxcl1, Tnf, Il1a, Il1b, and Hbegf expression levels were analyzed using qRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 by Student’s t test.

Article Snippet: Cell culture supernatant from keratinocytes was collected, and protein levels of IL-8 (BioLegend), CXCL5 (R&D Systems), CCL20 (Boster Immunoleader), and HB-EGF (Abcam) were measured by ELISA according to the manufacturer’s instructions.

Techniques: Staining, Immunostaining, Isolation, Expressing, Quantitative RT-PCR

Journal: Journal of Clinical Investigation

Article Title: MicroRNA-132 enhances transition from inflammation to proliferation during wound healing

doi: 10.1172/jci79052

Figure Lengend Snippet: Figure 9. Skin wound healing is impaired in miR-132–KO mice. (A) Representative images of wounds on the backs of miR-132–KO mice (n = 9) and their WT littermate controls (n = 6) on days 0, 2, 4, 6, and 8 after wounding. Wound closures were quantified and are presented as the percentage of the initial wound area size. (B) qRT-PCR was performed to detect Ccl20, Cxcl5, Cxcl1, Tnf, Il1a, and Il1b expression levels in the intact and wound-edge skin of both WT (n = 5) and KO (n = 9) mice 2 days after injury. *P < 0.05 and **P < 0.01 by Student’s t test. (C) H&E staining (left panels) and immunostaining of Gr-1 (mid- dle and right panels) in the wound area in WT and KO mice 2 days after injury. White arrows demarcate wound edges. Scale bars: 50 μm. (D) Chemotaxis of neutrophils isolated from the peripheral blood of WT (n = 5) and KO mice (n = 7) toward the medium containing 8 μM N-formylmethionyl-leucyl-phenyla- lanine (fMLP). Migrating cells were quantified by flow cytometry. Plots show the FSC/SSC of the migrated cells. (E) Immunostaining of Ki-67 and HB-EGF in wound-edge skin from WT (n = 9) and KO mice (n = 5) 2 days after injury. Sections were counterstained using DAPI (blue, nucleus). The number of Ki-67– positive cells was counted. (F) Immunostaining of p-STAT3 and p-ERK in wound-edge skin from WT (n = 9) and KO mice (n = 5) 2 days after injury. Scale bars: 50 μm (E and F). *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test.

Article Snippet: Cell culture supernatant from keratinocytes was collected, and protein levels of IL-8 (BioLegend), CXCL5 (R&D Systems), CCL20 (Boster Immunoleader), and HB-EGF (Abcam) were measured by ELISA according to the manufacturer’s instructions.

Techniques: Quantitative RT-PCR, Expressing, Staining, Immunostaining, Chemotaxis Assay, Isolation, Flow Cytometry

Journal: Journal of Clinical Investigation

Article Title: MicroRNA-132 enhances transition from inflammation to proliferation during wound healing

doi: 10.1172/jci79052

Figure Lengend Snippet: Figure 11. Inhibition of miR-132 delays reepithelialization of human ex vivo skin wounds. Human ex vivo skin wounds were treated topically with anti–miR-132 (n = 4) or anti–miR-Ctrl (n = 4) after injury, and miR-132 blocking efficiency was confirmed by qRT-PCR (A). (B) H&E staining of the ex vivo skin wounds 3 days after injury. Black arrows demarcate the initial wound edges, while the red arrows indicate a newly formed epidermis. Scale bars: 200 μm. (C) Immunostaining of Ki-67 in ex vivo skin wounds 3 days after injury. Sections were counterstained with DAPI. The number of Ki-67– positive cells was counted. White arrows demarcate the wound edges. Scale bars: 50 μm. CXCL1, CXCL5, CCL20 (D), and HB-EGF (E) expression levels were detected by qRT-PCR in ex vivo wounds treated with anti–miR-Ctrl (n = 4) or anti–miR-132 (n = 4) 3 days after injury. (F) Schematic summary of the regulation and function of miR-132 during skin wound healing. *P < 0.05 and **P < 0.01 by Student’s t test.

Article Snippet: Cell culture supernatant from keratinocytes was collected, and protein levels of IL-8 (BioLegend), CXCL5 (R&D Systems), CCL20 (Boster Immunoleader), and HB-EGF (Abcam) were measured by ELISA according to the manufacturer’s instructions.

Techniques: Inhibition, Ex Vivo, Blocking Assay, Quantitative RT-PCR, Staining, Immunostaining, Expressing