full length serpine1 construct  (Addgene inc)

Journal: Cancer cell international

Article Title: LncRNA H19 acts as a ceRNA to promote glioblastoma malignancy by sponging miR-19b-3p and upregulating SERPINE1.

doi: 10.1186/s12935-025-03868-x

Figure Lengend Snippet: Fig. 1 Schematic representation of the process used to identify an oncogene regulated by H19 based on total RNA-seq data (GSE243116). SERPINE1 was identified as a potential oncogene upregulated by H19 in GBM cell lines (p < 0.05).

Article Snippet: A pcDNA3.1 vector containing a fulllength H19 construct (pcDNA3.1-H19) and a pCMVSPORT6 vector containing a full-length SERPINE1 construct (pCMV-SPORT6-SERPINE1) were purchased from Addgene (Cambridge, MA, USA) and the Korea Human Gene Bank (Medical Genomics Research Center, KRIBB, Daejeon, Republic of Korea), respectively.

Techniques: RNA Sequencing

Journal: Cancer cell international

Article Title: LncRNA H19 acts as a ceRNA to promote glioblastoma malignancy by sponging miR-19b-3p and upregulating SERPINE1.

doi: 10.1186/s12935-025-03868-x

Figure Lengend Snippet: Fig. 3 Identification of miR-19b-3p as a tumor suppressor that binds to H19 and SERPINE1 in GBM cell lines. (A) miRNAs that bind to H19 and SERPINE1 were predicted using RNA interactome databases. (B) Overall survival analysis of miR-19b-3p (p = 0.004) in GBM patients was performed using the On coLnc database. (C) The miR-19b-3p sequence responsible for binding to H19 (H19-WT) and the mutant sequence of H19 (H19-MUT) were predicted using the ENCORI database. (D) The binding sequence responsible for miR-19b-3p binding to SERPINE1 (SERPINE1-WT) and the mutant sequence of SERPINE1 (SERPINE1-MUT) were also predicted using the ENCORI database. (E) The binding activities of miR-19b-3p mimic with H19-WT or H19-MUT were evaluated using a dual-luciferase reporter assay. (F) The binding activities of miR-19b-3p mimic with SERPINE1-WT or SERPINE1-MUT were also evaluated using a dual-luciferase reporter assay. **p < 0.01, ***p < 0.001

Article Snippet: A pcDNA3.1 vector containing a fulllength H19 construct (pcDNA3.1-H19) and a pCMVSPORT6 vector containing a full-length SERPINE1 construct (pCMV-SPORT6-SERPINE1) were purchased from Addgene (Cambridge, MA, USA) and the Korea Human Gene Bank (Medical Genomics Research Center, KRIBB, Daejeon, Republic of Korea), respectively.

Techniques: Sequencing, Binding Assay, Mutagenesis, Luciferase, Reporter Assay

Journal: Cancer cell international

Article Title: LncRNA H19 acts as a ceRNA to promote glioblastoma malignancy by sponging miR-19b-3p and upregulating SERPINE1.

doi: 10.1186/s12935-025-03868-x

Figure Lengend Snippet: Fig. 6 A schematic diagram of the ceRNA network illustrating the interaction between H19, miR-19b-3p, and SERPINE1. In this network, H19 competes with SERPINE1 mRNA for binding to miR-19b-3p. When H19 acts as a ceRNA sponging miR-19b-3p, the binding of miR-19b-3p to SERPINE1 mRNA is reduced. This reduction alleviates the inhibition of SERPINE1 translation by miR-19b-3p, ultimately leading to the upregulation of SERPINE1 in response to increased H19 expression

Article Snippet: A pcDNA3.1 vector containing a fulllength H19 construct (pcDNA3.1-H19) and a pCMVSPORT6 vector containing a full-length SERPINE1 construct (pCMV-SPORT6-SERPINE1) were purchased from Addgene (Cambridge, MA, USA) and the Korea Human Gene Bank (Medical Genomics Research Center, KRIBB, Daejeon, Republic of Korea), respectively.

Techniques: Binding Assay, Inhibition, 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: Expression plasmids encoding the precursor ectodomain of human SERPINE1 (SERPINE1-bio-His; Addgene plasmid no. 52077; http://n2t.net/addgene:52077 ; RRID:Addgene_52077), SERPINA1 (SERPINA-bio-His; Addgene plasmid no. 52182; http://n2t.net/addgene:52182 ; RRID:Addgene_52182), and SERPINC1 (SERPINC1-bio-His; Addgene plasmid no. 52076; http://n2t.net/addgene:52076 ; RRID:Addgene_52076) were a kind gift of Gavin Wright ( ).

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: Expression plasmids encoding the precursor ectodomain of human SERPINE1 (SERPINE1-bio-His; Addgene plasmid no. 52077; http://n2t.net/addgene:52077 ; RRID:Addgene_52077), SERPINA1 (SERPINA-bio-His; Addgene plasmid no. 52182; http://n2t.net/addgene:52182 ; RRID:Addgene_52182), and SERPINC1 (SERPINC1-bio-His; Addgene plasmid no. 52076; http://n2t.net/addgene:52076 ; RRID:Addgene_52076) were a kind gift of Gavin Wright ( ).

Techniques: RNA Sequencing, Expressing, Infection

Journal: bioRxiv

Article Title: Senescence of stromal cells contributes to endometrium dysfunction and embryo implantation failure

doi: 10.1101/2021.07.19.452880

Figure Lengend Snippet: A Intracellular ROS estimation by DCF fluorescence intensity (n = 3). B Dynamics of intracellular H 2 O 2 levels in young and senescent ESCs in course of decidualization assessed by Hyper/SypHer ratios (n = 3). C Amounts of H 2 O 2 excreted into the intracellular space by young and senescent ESCs (n = 3). D Hyper/SypHer ratios in Hyper/SypHer-expressing ESCs co-cultured with young or senescent cells reflecting H 2 O 2 transmitting (n = 3). E Decreased intracellular ROS levels in ESCs upon tempol treatment estimated by DCF fluorescence intensity (n = 3). F Fluorescence intensity of the decidual reporter protein in undifferentiated and decidualized ESCs co-cultured with young, senescent, or tempol-pretreated senescent cells (n = 3). G Quantification of invasion areas of BeWo spheroids into the monolayers of decidualizaing young, senescent or tempol-pretreated senescent ESCs (n = 30 spheroids for each condition). H, I Verification of CRISPR-Cas9 mediated SERPINE1 knockout in ESCs performed by Western blotting (H) and RT-PCR (I) (n = 3). J Quantification of invasion areas of BeWo spheroids into the monolayers of decidualizaing senescent ESCs with or without SERPINE1 knockout (n = 30 spheroids for each condition). K, L Verification of CRISPR-Cas9 mediated SERPINE1 overexpression in ESCs performed by Western blotting (K) and RT-PCR (L) (n = 3). M Quantification of invasion areas of BeWo spheroids into the monolayers of decidualizaing ESCs with or without SERPINE1 overexpression (n = 30 spheroids for each condition). Data information: In (A–F), (I) and (L), data are presented as mean ± SD. In (G) and (M) data are presented as median ± IQR. *P<0.05, **P<0.01, ***P<0.005 vs senescent cells, #P<0.05, ##P<0.01 vs young cells (A), (B), (F), (D) (ANOVA with Tukey HSD), (C), (D), (E), (I), (J), (L), (M) (Student’s t-test).

Article Snippet: For CRISPR-mediated SERPINE1 knockout and transactivation the following lentivectors were used: pCC_01 - hU6-BsmBI-sgRNA(E+F)-barcode-EFS-Cas9-NLS-2A-Puro-WPRE (Addgene 139086, ) and pCC_05 - hU6-BsmBI-sgRNA(E+F)-barcode-EFS-dCas9-NLS-VPR-2A-Puro-WPRE (Addgene 139090, ).

Techniques: Fluorescence, Expressing, Cell Culture, CRISPR, Knock-Out, Western Blot, Reverse Transcription Polymerase Chain Reaction, Over Expression

Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

Article Title: PAI-1 is a novel component of the miR-17~92 signaling that regulates pulmonary artery smooth muscle cell phenotypes

doi: 10.1152/ajplung.00137.2017

Figure Lengend Snippet: Primer sequences

Article Snippet: PAI-1 overexpression (PAI-OE) plasmids (PAI1-Bio-His, or Serpine1-bio-His) were purchased from Addgene (Cambridge, MA) and transfected into human PASMCs using Lipofectamine 2000.

Techniques: Luciferase, Plasmid Preparation, Cloning