Journal: iScience

Article Title: Evolutionary conserved regulation of TFEB stability by the E3 ubiquitin ligase WWP2 modulates response to stress in vivo

doi: 10.1016/j.isci.2025.111838

Figure Lengend Snippet:

Article Snippet: recombinant human 6His-Ube1 , RD System , Cat# E-305.

Techniques: Virus, Recombinant, Ubiquitin Proteomics, Luciferase, Mutagenesis, Reverse Transcription, SYBR Green Assay, Plasmid Preparation, Software, Lysis

Journal: Cell death discovery

Article Title: Endothelial senescence induced by PAI-1 promotes endometrial fibrosis.

doi: 10.1038/s41420-025-02377-0

Figure Lengend Snippet: Fig. 3 PAI-1 derived from endometrial stromal cells promotes senescence of ECs. A Analysis of interactions between various cells. B Analysis of interactions between PAI-1 + ESC and other various cells. C Immunofluorescence staining of PAI-1 (green) and vessels (red) in IUA patients (n = 5). scale bar = 100 μm. The supernatant from PAI-1+ ESCs was collected to stimulate HUVECs. D–J. Subsequently, the mRNA levels of P16 and P21 were analyzed by qPCR (n = 12) (D); the protein level of P21 was assessed by western blotting (n = 3) (E); the mRNA and protein levels of IL-6 were examined by qPCR (n = 6) and western blotting (n = 3), respectively (F); and the protein level of eNOS was determined by western blotting (n = 3) (G). SA-β-gal staining was observed (n = 3). scale bar = 100 μm (H), HUVECs migration was assayed (n = 3). scale bar = 100 μm (I), and tube formation was examined (n = 3) (J). scale bar = 100 μm.

Article Snippet: The recombinant human PAI1 (R&D Systems, cat: 1786-PI-010, USA) was dissolved in the culture medium and subsequently administered to the cells for a duration of 60 h. For the preparation of endometrial stromal cells (ESCs) [6, 43], fresh endometrial tissues were cut into small fragments and digested with a mixture of collagenase type I (Sigma, C2674, USA), hyaluronidase (Sigma, H3506, USA), and DNase (Roche, 10104159001, Switzerland) at 37 °C.

Techniques: Derivative Assay, Staining, Western Blot, Migration

Journal: Cell death discovery

Article Title: Endothelial senescence induced by PAI-1 promotes endometrial fibrosis.

doi: 10.1038/s41420-025-02377-0

Figure Lengend Snippet: Fig. 4 PAI-1 promotes endothelial cell senescence through uPAR. A Immunofluorescence staining of uPAR (green) and CD31 (red) in endometria from IUA patients (n = 12) and controls (n = 12). Quantized co-expression area (%) was showed at right. scale bar = 100 μm. B, C The mRNA (n = 12) and protein levels (n = 3) of uPA in PAI-1+ESCs. D, E The mRNA (n = 11) and protein levels (n = 3) of uPAR in HUVECs treated with PAI-1+ESCs supernatant. F, G The mRNA and protein levels of P21 (n = 3) in HUVECs treated with Dox and transfected with si- PLAUR.

Article Snippet: The recombinant human PAI1 (R&D Systems, cat: 1786-PI-010, USA) was dissolved in the culture medium and subsequently administered to the cells for a duration of 60 h. For the preparation of endometrial stromal cells (ESCs) [6, 43], fresh endometrial tissues were cut into small fragments and digested with a mixture of collagenase type I (Sigma, C2674, USA), hyaluronidase (Sigma, H3506, USA), and DNase (Roche, 10104159001, Switzerland) at 37 °C.

Techniques: Staining, Expressing, Transfection

Journal: Cell death discovery

Article Title: Endothelial senescence induced by PAI-1 promotes endometrial fibrosis.

doi: 10.1038/s41420-025-02377-0

Figure Lengend Snippet: Fig. 5 TGF-β upregulates PAI-1 in SMAD dependent manner. A The mRNA levels of PAI-1, ACTA2, and COL1A1 were examined by qPCR in ESCs after treatment with 10 ng/ml TGF-β for 3, 6, 12, 24 h respectively (n = 3). B The protein levels of PAI-1, ACTA2, and COL1A1 were tested by western blotting in ESCs after treatment with 10 ng/ml TGF-β for 6, 12, 24 h respectively (n = 3). C The mRNA level of PAI-1 were examined by qPCR in ESCs after treatment with 10 ng/ml TGF-β with or without SB-421542 (n = 3). D The protein levels of PAI-1, p-SMAD2, and SMAD2 were tested by western blotting in ESCs after treatment with 10 ng/ml TGF-β with or without SB-421542 (n = 3).

Article Snippet: The recombinant human PAI1 (R&D Systems, cat: 1786-PI-010, USA) was dissolved in the culture medium and subsequently administered to the cells for a duration of 60 h. For the preparation of endometrial stromal cells (ESCs) [6, 43], fresh endometrial tissues were cut into small fragments and digested with a mixture of collagenase type I (Sigma, C2674, USA), hyaluronidase (Sigma, H3506, USA), and DNase (Roche, 10104159001, Switzerland) at 37 °C.

Techniques: Western Blot

Journal: Cell death discovery

Article Title: Endothelial senescence induced by PAI-1 promotes endometrial fibrosis.

doi: 10.1038/s41420-025-02377-0

Figure Lengend Snippet: Fig. 6 PAI-1 inhibitors can inhibit endothelial senescence and endometrial fibrosis in mice. A Masson’s trichrome staining and immunohistochemical staining for COL1A1, α-SMA, PAI-1, uPA, and uPAR were performed on endometrial samples from normal controls, both with and without TPX, as well as in the IUA mouse model, both with and without TPX (n = 6). scale bar = 100 μm. B Immunofluorescence staining was conducted to detect P21 (green) and CD31 (red) in endometrial samples from normal controls, both with and without TPX, and in the IUA mouse model, both with and without TPX (n = 6). scale bar = 100 μm. The quantized results analyzed by ImageJ showed on right, each bar represents the mean ± SEM. ANOVA test, **p < 0.001, ***p < 0.0001.

Article Snippet: The recombinant human PAI1 (R&D Systems, cat: 1786-PI-010, USA) was dissolved in the culture medium and subsequently administered to the cells for a duration of 60 h. For the preparation of endometrial stromal cells (ESCs) [6, 43], fresh endometrial tissues were cut into small fragments and digested with a mixture of collagenase type I (Sigma, C2674, USA), hyaluronidase (Sigma, H3506, USA), and DNase (Roche, 10104159001, Switzerland) at 37 °C.

Techniques: Staining, Immunohistochemical staining

Journal: Cell death discovery

Article Title: Endothelial senescence induced by PAI-1 promotes endometrial fibrosis.

doi: 10.1038/s41420-025-02377-0

Figure Lengend Snippet: Fig. 7 Schematic diagram illustrating how PAI-1 derived from ESCs promotes endothelial aging, leading to endometrial fibrosis. Trauma or infection elevates TGF-β in the endometrial microenvironment. TGF-β promotes the expression of PAI-1 in ESCs by activating SMAD2. PAI-1 synergizes with uPA to act on the uPAR receptor on endothelial cells, accelerating cellular senescence. Following endothelial cell senescence, the cells’ angiogenic capacity is inhibited, and they contribute to endometrial fibrosis by releasing factors such as SASP.

Article Snippet: The recombinant human PAI1 (R&D Systems, cat: 1786-PI-010, USA) was dissolved in the culture medium and subsequently administered to the cells for a duration of 60 h. For the preparation of endometrial stromal cells (ESCs) [6, 43], fresh endometrial tissues were cut into small fragments and digested with a mixture of collagenase type I (Sigma, C2674, USA), hyaluronidase (Sigma, H3506, USA), and DNase (Roche, 10104159001, Switzerland) at 37 °C.

Techniques: Derivative Assay, Infection, Expressing

Journal: mBio

Article Title: Serine Protease Inhibitors Restrict Host Susceptibility to SARS-CoV-2 Infections

doi: 10.1128/mbio.00892-22

Figure Lengend Snippet: The investigated serpins reduce SARS-CoV-2 infection by inhibition of TMPRSS2-mediated spike protein cleavage. (A) HEK-293T cells transfected with the indicated expression plasmids for 24 h were infected with SARS-CoV-2 (MOI = 0.1) for 6 h, and viral RNA was measured by qPCR. (B and C) Posttransfection (24 h) HEK293T cells were infected for 2 h (MOI = 1) and then trypsinized, washed with PBS, and lysed, and the RNA was extracted. Levels of viral RNA were quantified from cDNA synthesized with (B) random hexamers (C) or only the forward primer selectively quantifying the negative sense RNA. Data are cumulative of three independent experiments performed in triplicate; mean and SEM are shown, and statistical significance was calculated by unpaired t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (D) Surface plasmon resonance analysis of TMPRSS2 binding to individual serpins. A 2-fold dilution series of TMPRSS2 ranging from 125 nM down to 7.8 nM over immobilized SERPINE1 with results shown as response units (RU). Binding kinetics for all serpins are summarized to the right, including the natural target for SERPINE1, tissue plasminogen activator (tPA), as a positive control. (E) TMPRSS2-mediated S-protein cleavage in the presence or absence of individual serpins and the known protease inhibitor nafamostat mesylate. Data from three independent experiments were quantified, and a representative blot is shown. (F) The intensity of bands in panel E corresponding to cleaved S-protein was quantified using ImageJ (Fuji) and normalized to S-protein and TMPRSS2 control. Mean values and SEM are shown; statistical significance was calculated by unpaired t test (*, P < 0.05; **, P < 0.01). (G) HBEC ALI cultures were preincubated apically with recombinant SERPINE1, SERPINA1, or SERPINC1 and infected with SARS-CoV-2 at an MOI of 0.05. The accumulated viral release from the apical side was quantified by qPCR at the indicated time points ( n = 3). Mean and SEM are shown; statistical significance was calculated by unpaired t test (*, P < 0.05; **, P < 0.01). (H) The concentrations of apically released SERPINA1 and SERPINE2 from HBEC ALI cultures from both group high and group low were determined by ELISA. The apical secretions were collected at three time points ( n = 3). Mean and SEM are shown; statistical significance was calculated by unpaired t test (***, P < 0.001).

Article Snippet: Recombinant SERPINE1 protein (PAI-1, 1786-PI), SERPINA1 protein (A1AT, 1268-PI), SERPINC1 protein (ATIII, 1267-PI), and nafamostat mesylate (3081) were purchased from R&D Systems.

Techniques: Infection, Inhibition, Transfection, Expressing, Synthesized, SPR Assay, Binding Assay, Positive Control, Protease Inhibitor, Control, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: mBio

Article Title: Serine Protease Inhibitors Restrict Host Susceptibility to SARS-CoV-2 Infections

doi: 10.1128/mbio.00892-22

Figure Lengend Snippet: Single-cell RNA sequencing shows distinct expression patterns of serpins in primary lung cells. (A) Schematic illustration of cell types annotated in the single-cell RNA analysis of HBEC ALI cultures. (B) UMAP plot (bidimensional), colored by annotated cell clusters. (C) Coarse-grained graph showing mean cell cluster group expression for SERPINA1, SERPINE1, and SERPINE2. (D) Infection levels analyzed by qPCR and shown as fold increase over 12-h input. (E) Heatmap displaying the mean expression of SERPINA1, SERPINE1, and SERPINE2 upon SARS-CoV-2 infection (±, MOI = 0.05) in HBEC ALI cultures from a group high (H) and a group low (L) donor.

Article Snippet: Recombinant SERPINE1 protein (PAI-1, 1786-PI), SERPINA1 protein (A1AT, 1268-PI), SERPINC1 protein (ATIII, 1267-PI), and nafamostat mesylate (3081) were purchased from R&D Systems.

Techniques: RNA Sequencing, Expressing, Infection

Journal: Open Biology

Article Title: Phosphorylation of Parkin at Serine65 is essential for activation: elaboration of a Miro1 substrate-based assay of Parkin E3 ligase activity

doi: 10.1098/rsob.130213

Figure Lengend Snippet: PINK1-dependent phosphorylation of Parkin Ser 65 leads to activation of Parkin E3 ligase activity and multi-monoubiquitylation of Miro1. Wild-type (WT) ( a ) but not kinase-inactive (KI) ( b ) PINK1 activates wild-type Parkin E3 ligase activity leading to Miro1 multi-monoubiquitylation, an effect that is blocked by mutant Parkin Ser65Ala (S65A) ( c ). Two micrograms of wild-type or S65A Parkin were incubated with indicated amounts of wild-type or kinase-inactive (D359A) MBP-TcPINK in a kinase reaction (50 mM Tris–HCl (pH 7.5), 0.1 mM ethylene glycol tetra-acetic acid (EGTA), 10 mM MgCl 2 , 0.1% 2-mercaptoethanol and 0.1 mM ATP) for 60 min. The ubiquitylation reaction was then initiated by addition of ubiquitylation assay components (50 mM Tris–HCl (pH 7.5), 0.05 mM EGTA, 10 mM MgCl 2 , 0.5% 2-mercaptoethanol, 0.12 μM human recombinant E1 purified from Sf21 insect cell line, 1 μM human recombinant UbcH7 purified from E. coli , 0.05 mM Flag-ubiquitin (Boston Biochem) and 2 mM ATP) and 2 μg of His-Sumo-Miro1. Reactions were terminated after 60 min by addition of SDS–PAGE loading buffer and resolved by SDS–PAGE. Miro1, ubiquitin, Parkin and PINK1 were detected using anti-SUMO, anti-FLAG, anti-Parkin and anti-MBP antibodies, respectively. Representative of three independent experiments.

Article Snippet: Kinase assays were incubated at 30°C for 60 min followed by addition of ubiquitylation assay components and Mastermix to a final volume of 50 μl (50 mM Tris–HCl (pH 7.5), 0.05 mM EGTA, 10 mM MgCl 2 , 0.5% 2-mercaptoethanol, 0.12 μM human recombinant E1 purified from Sf21 insect cell line, 1 μM human recombinant UbcH7 and 2 μg 6xHis-Sumo-Miro1 (wild-type or point mutants) both purified from E. coli , 0.05 mM Flag-ubiquitin (Boston Biochem) and 2 mM ATP).

Techniques: Activation Assay, Activity Assay, Mutagenesis, Incubation, Ubiquitin Assay, Recombinant, Purification, SDS Page

Journal: Developmental cell

Article Title: Ari-1 regulates myonuclear organization together with Parkin and is associated with aortic aneurysms

doi: 10.1016/j.devcel.2018.03.020

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: A 50 μL reaction mixture for KoiU2 ubiquitination contained 1μM human E1 recombinant protein (Enzo), 5μM UbcH7 (Boston Biochem), 2μM GST-tagged E3 ubiquitin ligase, 5μM T7-KoiU2, 5mM Mg-ATP (Enzo) 2.5μM biotinylated ubiquitin (Enzo) and 1mM DTT in 1× ubiquitination buffer (Enzo).

Techniques: Recombinant, Plasmid Preparation, Protease Inhibitor, Lysis, Transfection, SYBR Green Assay, Mutagenesis, Modification, shRNA