Journal: Frontiers in Immunology

Article Title: Urokinase-type plasminogen activator and plasminogen activator inhibitor-1 complex as a serum biomarker for COVID-19

doi: 10.3389/fimmu.2023.1299792

Figure Lengend Snippet: Free, non-complexed PAI-1 and uPA/PAI-1 complex levels in circulation are highest in severely ill COVID-19 patients. (A) Active PAI-1 (noncomplexed) levels were measured in patients and healthy donor serum using functional ELISA. Western blot analysis was used for serum level assessment of non-complexed PAI-1 antigen (B) with a molecular weight of 45 kDa, tPA (C) with a molecular weight of 70 kDa, tPA/PAI-1 complex with a molecular weight of 120 kDa (D) , uPA (E, F) with a molecular weight of 50 kDa, and uPA/PAI-1 (E, G) complexes detectable as 100 Da band. (A–D, F–G) The quantification of band intensity of single proteins or protein complexes normalized to a sample from a healthy donor is given. (H, I) The Spearman coefficient r was used to correlate the interrelation of PAs/PAI-1 vs. plasmin/a2-antiplasmin (n = 36 per group). *p < 0.1; **p < 0.05; ***p < 0.01; n/s – not significant.

Article Snippet: PAI-1 rabbit polyclonal antibodies (1:1000, #ab66705, Abcam, Cambridge, UK); tPA rabbit polyclonal antibodies (1:1000, #sc-15346, Santa Cruz, Dallas, USA); uPA rabbit polyclonal antibodies (1:1000, #17968-1-AP, Proteintech, San Diego, USA); uPAR rabbit polyclonal antibodies (1 ug/ml) ( ); Plg rabbit polyclonal antibodies (0,5 ug/ml) ( ); TGFβ1 rabbit polyclonal antibodies (1:1000, #sc-146, Santa Cruz, Dallas, USA); TNFα rabbit polyclonal antibodies (Cell Signaling #3707S, 1:1000); VCAM1 rabbit polyclonal antibodies (1:1000, #sc8304, Santa Cruz, Dallas, USA); α2-antiplasmin rabbit polyclonal antibodies (1:1000, #sc73659, Santa Cruz, Dallas, USA); IL1β hamster polyclonal were kindly provided by members of the FACS Core Facility of Juntendo University (1 ug/ml); secondary rabbit anti hamster HRP-conjugated (1:2000, #ab5745, Abcam, Cambridge, UK); secondary goat anti-rabbit polyclonal HRP-conjugated (1:8000, #sc2004, Santa Cruz, Dallas, USA).

Techniques: Functional Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Molecular Weight

Journal: Frontiers in Immunology

Article Title: Urokinase-type plasminogen activator and plasminogen activator inhibitor-1 complex as a serum biomarker for COVID-19

doi: 10.3389/fimmu.2023.1299792

Figure Lengend Snippet: Alterations of endothelial dysfunction serum markers in COVID-19 patients related to plasmin activity. Serum soluble VCAM-1 (sVACM) (A) and uPAR (B) were measured using Western blot analysis, and the quantification of band intensity normalized to a sample from a healthy donor was given. The Spearman coefficient r was used for the correlation analysis of serum sVCAM1 and suPAR against PAI-1 activity (n = 45 per group) and non-complexed PAI-1 antigen (n = 36 per group) (C, D) . Another Spearman correlation was done for sVCAM1 and suPAR against plasmin/α2AP) (Pm/α2AP) (n = 36 per group) (E, F) . N depicts the number of patients tested in each group. *p < 0.1; **p < 0.05; ***p < 0.01; n/s – not significant .

Article Snippet: PAI-1 rabbit polyclonal antibodies (1:1000, #ab66705, Abcam, Cambridge, UK); tPA rabbit polyclonal antibodies (1:1000, #sc-15346, Santa Cruz, Dallas, USA); uPA rabbit polyclonal antibodies (1:1000, #17968-1-AP, Proteintech, San Diego, USA); uPAR rabbit polyclonal antibodies (1 ug/ml) ( ); Plg rabbit polyclonal antibodies (0,5 ug/ml) ( ); TGFβ1 rabbit polyclonal antibodies (1:1000, #sc-146, Santa Cruz, Dallas, USA); TNFα rabbit polyclonal antibodies (Cell Signaling #3707S, 1:1000); VCAM1 rabbit polyclonal antibodies (1:1000, #sc8304, Santa Cruz, Dallas, USA); α2-antiplasmin rabbit polyclonal antibodies (1:1000, #sc73659, Santa Cruz, Dallas, USA); IL1β hamster polyclonal were kindly provided by members of the FACS Core Facility of Juntendo University (1 ug/ml); secondary rabbit anti hamster HRP-conjugated (1:2000, #ab5745, Abcam, Cambridge, UK); secondary goat anti-rabbit polyclonal HRP-conjugated (1:8000, #sc2004, Santa Cruz, Dallas, USA).

Techniques: Activity Assay, Western Blot

Journal: Frontiers in Immunology

Article Title: Urokinase-type plasminogen activator and plasminogen activator inhibitor-1 complex as a serum biomarker for COVID-19

doi: 10.3389/fimmu.2023.1299792

Figure Lengend Snippet: Correlation of fibrinolytic and inflammation markers in COVID-19 patients. (A) Heatmap of Spearman correlation coefficient. Pairs with positive coefficients are in brown, and those with negative coefficients are in blue. (B) P values associated with Spearman’s correlation of different parameters. (C) Heat map of Pearson’s correlation coefficient biomarkers of COVID-19 patients. The correlation coefficients are represented in the orange/blue color intensity change, shown in the color bar, whereby variables indicated by green or orange were positively or negatively correlated, respectively. Correlations were statistically significant (p<0.05) with 95% CIs for each correlation coefficient. (D) Model demonstrating the differences between COVID-19 patients in the uncomplicated (right side) and complicated (left side) phases. PAI-1, plasminogen activator inhibitor1); tPA, tissue-type plasminogen activator; uPA, urokinase plasminogen activator; SARS-CoV2, Severe acute respiratory syndrome coronavirus 2.

Article Snippet: PAI-1 rabbit polyclonal antibodies (1:1000, #ab66705, Abcam, Cambridge, UK); tPA rabbit polyclonal antibodies (1:1000, #sc-15346, Santa Cruz, Dallas, USA); uPA rabbit polyclonal antibodies (1:1000, #17968-1-AP, Proteintech, San Diego, USA); uPAR rabbit polyclonal antibodies (1 ug/ml) ( ); Plg rabbit polyclonal antibodies (0,5 ug/ml) ( ); TGFβ1 rabbit polyclonal antibodies (1:1000, #sc-146, Santa Cruz, Dallas, USA); TNFα rabbit polyclonal antibodies (Cell Signaling #3707S, 1:1000); VCAM1 rabbit polyclonal antibodies (1:1000, #sc8304, Santa Cruz, Dallas, USA); α2-antiplasmin rabbit polyclonal antibodies (1:1000, #sc73659, Santa Cruz, Dallas, USA); IL1β hamster polyclonal were kindly provided by members of the FACS Core Facility of Juntendo University (1 ug/ml); secondary rabbit anti hamster HRP-conjugated (1:2000, #ab5745, Abcam, Cambridge, UK); secondary goat anti-rabbit polyclonal HRP-conjugated (1:8000, #sc2004, Santa Cruz, Dallas, USA).

Techniques:

Journal: The FASEB Journal

Article Title: Serpin family E member 1 enhances myometrium contractility by increasing ATP production during labor

doi: 10.1096/fj.202301804r

Figure Lengend Snippet: FIGURE 1 SERPINE1 is upregulated in human in-labor myometrium. (A) Heat map of differentially expressed mRNAs in human in- labor and non-labor myometrium. (B) Volcano plot of all proteins alteration in in-labor myometrium, compared to non-labor myometrium. Red dots represent statistically significant upregulated genes. Blue dots represent statistically significant downregulated genes. (C) The mRNA expression levels of SERPINE1 in myometrium.(D, E) The protein expression levels of SERPINE1 in myometrium. (F) Protein spatial structure of SERPINE1. (G, H) Immunofluorescence staining of SERPINE1+ (green) and α-SMA+ (red) in myometrium (G) and the quantification for the relative SERPINE1+ positive uterine smooth muscle cell immunoreactivity (H), IgG was used as negative controls. Data are expressed as mean ± SD, *p < .05; **p < .01.

Article Snippet: After blocking with 10% goat serum at 37°C for 1 h, the sections were stained at 4°C overnight with either rabbit polyclonal SERPINE1 antibody (1:200, NBP1- 19773, NOVUS), mouse polyclonal α- SMA (1:300, ab7817, Abcam), rabbit polyclonal ATP5PF antibody (1:1000, A3751, ABclonal), rabbit monoclonal SERPINE1 antibody (1:30, ab222754, Abcam), mouse monoclonal TOMM20 (1:100, ab7817, Abcam) followed by Alexa Fluor 647 Goat anti- mouse secondary antibody (1:500, ab15011, Abcam) or goat anti- rabbit Alexa Fluor 488- IgG (1:500, ab150077, Abcam).

Techniques: Expressing, Immunofluorescence, Staining

Journal: The FASEB Journal

Article Title: Serpin family E member 1 enhances myometrium contractility by increasing ATP production during labor

doi: 10.1096/fj.202301804r

Figure Lengend Snippet: FIGURE 2 SERPINE1 contributes to hypoxia driven contraction in myometrium. (A) Recordings of isometric tension measurement of myometrium. (B, C) AUC (B) and amplitude (C) of contractions over time of myometrium. Data are expressed as mean ± SD. */#p < .05, **/##p < .01, compared to normoxia group.

Article Snippet: After blocking with 10% goat serum at 37°C for 1 h, the sections were stained at 4°C overnight with either rabbit polyclonal SERPINE1 antibody (1:200, NBP1- 19773, NOVUS), mouse polyclonal α- SMA (1:300, ab7817, Abcam), rabbit polyclonal ATP5PF antibody (1:1000, A3751, ABclonal), rabbit monoclonal SERPINE1 antibody (1:30, ab222754, Abcam), mouse monoclonal TOMM20 (1:100, ab7817, Abcam) followed by Alexa Fluor 647 Goat anti- mouse secondary antibody (1:500, ab15011, Abcam) or goat anti- rabbit Alexa Fluor 488- IgG (1:500, ab150077, Abcam).

Techniques:

Journal: The FASEB Journal

Article Title: Serpin family E member 1 enhances myometrium contractility by increasing ATP production during labor

doi: 10.1096/fj.202301804r

Figure Lengend Snippet: FIGURE 3 SERPINE1 is involved in hypoxia driven contraction in hMSMCs. (A) The mRNA expression levels of SERPINE1 in hMSMCs under hypoxic conditions or transferred with si-SERPINE1. (B–D) The protein expression of SERPINE1 in hMSMCs under hypoxia conditions or transferred with si-SERPINE1 were detected by western blot (B) and immunofluorescence staining (C, D). (E, F) Gel contraction (E) and the gel areas (F) of hMSMCs under hypoxic conditions or transferred with si-SERPINE1. Data are expressed as mean ± SD, *p < .05, **p < .01.

Article Snippet: After blocking with 10% goat serum at 37°C for 1 h, the sections were stained at 4°C overnight with either rabbit polyclonal SERPINE1 antibody (1:200, NBP1- 19773, NOVUS), mouse polyclonal α- SMA (1:300, ab7817, Abcam), rabbit polyclonal ATP5PF antibody (1:1000, A3751, ABclonal), rabbit monoclonal SERPINE1 antibody (1:30, ab222754, Abcam), mouse monoclonal TOMM20 (1:100, ab7817, Abcam) followed by Alexa Fluor 647 Goat anti- mouse secondary antibody (1:500, ab15011, Abcam) or goat anti- rabbit Alexa Fluor 488- IgG (1:500, ab150077, Abcam).

Techniques: Expressing, Western Blot, Immunofluorescence, Staining

Journal: The FASEB Journal

Article Title: Serpin family E member 1 enhances myometrium contractility by increasing ATP production during labor

doi: 10.1096/fj.202301804r

Figure Lengend Snippet: FIGURE 4 SERPINE1 is involved in metabolism in hMSMCs under hypoxia. (A) Volcano plot of all genes alteration in SERPINE1- knockdown hMSMCs under hypoxia, compared to control group. Red dots represent statistically significant upregulated genes. Blue dots represent statistically significant downregulated genes. (B) Bubble diagram of partial significantly enriched pathways for deferential expressed genes in SERPINE1-knockdown hMSMCs under hypoxia. (C, D) Bar diagram of partial biology progress GO term clusters for upregulated genes (C) and downregulated (D) genes in SERPINE1-knockdown hMSMCs under hypoxia.

Article Snippet: After blocking with 10% goat serum at 37°C for 1 h, the sections were stained at 4°C overnight with either rabbit polyclonal SERPINE1 antibody (1:200, NBP1- 19773, NOVUS), mouse polyclonal α- SMA (1:300, ab7817, Abcam), rabbit polyclonal ATP5PF antibody (1:1000, A3751, ABclonal), rabbit monoclonal SERPINE1 antibody (1:30, ab222754, Abcam), mouse monoclonal TOMM20 (1:100, ab7817, Abcam) followed by Alexa Fluor 647 Goat anti- mouse secondary antibody (1:500, ab15011, Abcam) or goat anti- rabbit Alexa Fluor 488- IgG (1:500, ab150077, Abcam).

Techniques: Knockdown, Control

Journal: The FASEB Journal

Article Title: Serpin family E member 1 enhances myometrium contractility by increasing ATP production during labor

doi: 10.1096/fj.202301804r

Figure Lengend Snippet: FIGURE 5 SERPINE1 is involved in aerobic respiration in hMSMCs. (A) The ATP levels of SERPINE1 knockdown or overexpression hMSMCs under hypoxia. (B, C) ECAR of hMSMCs under hypoxia in response to glucose, oligomycin, and 2-DG. (D, E) OCR of hMSMCs under hypoxia in response to oligomycin, FCCP and Rot/AA. (F, G) OCR of SERPINE1 knockdown or overexpression hMSMCs under hypoxia in response to oligomycin, FCCP and Rot/AA. Data are expressed as mean ± SD, *p < .05, **p < .01. FCCP, fluorocarbonyl cyanide phenylhydrazone; OCR, Oxygen consumption rate; Res., respiration; Rot/AA, rotenone/antimycin A.

Article Snippet: After blocking with 10% goat serum at 37°C for 1 h, the sections were stained at 4°C overnight with either rabbit polyclonal SERPINE1 antibody (1:200, NBP1- 19773, NOVUS), mouse polyclonal α- SMA (1:300, ab7817, Abcam), rabbit polyclonal ATP5PF antibody (1:1000, A3751, ABclonal), rabbit monoclonal SERPINE1 antibody (1:30, ab222754, Abcam), mouse monoclonal TOMM20 (1:100, ab7817, Abcam) followed by Alexa Fluor 647 Goat anti- mouse secondary antibody (1:500, ab15011, Abcam) or goat anti- rabbit Alexa Fluor 488- IgG (1:500, ab150077, Abcam).

Techniques: Knockdown, Over Expression

Journal: The FASEB Journal

Article Title: Serpin family E member 1 enhances myometrium contractility by increasing ATP production during labor

doi: 10.1096/fj.202301804r

Figure Lengend Snippet: FIGURE 7 ATP5PF is involved in ATP production but has no effect on SERPINE1 expression in hMSMCs. (A) The expressions of SERPINE1 and ATP5PF in hMSMCs transferred with si-ATP5PF or ATP5PF overexpression lentiviral particles under hypoxia. (B) The ATP levels of ATP5PF knockdown or overexpression hMSMCs under hypoxia. (C, D) OCR of hMSMCs transferred with si-ATP5PF or ATP5PF overexpression lentiviral particles under hypoxia in response to oligomycin, FCCP and Rot/AA. Data are expressed as mean ± SEM, *p < .05, **p < .01. FCCP, fluorocarbonyl cyanide phenylhydrazone; OCR, oxygen consumption rate; Res., respiration; Rot/AA, rotenone/antimycin A.

Article Snippet: After blocking with 10% goat serum at 37°C for 1 h, the sections were stained at 4°C overnight with either rabbit polyclonal SERPINE1 antibody (1:200, NBP1- 19773, NOVUS), mouse polyclonal α- SMA (1:300, ab7817, Abcam), rabbit polyclonal ATP5PF antibody (1:1000, A3751, ABclonal), rabbit monoclonal SERPINE1 antibody (1:30, ab222754, Abcam), mouse monoclonal TOMM20 (1:100, ab7817, Abcam) followed by Alexa Fluor 647 Goat anti- mouse secondary antibody (1:500, ab15011, Abcam) or goat anti- rabbit Alexa Fluor 488- IgG (1:500, ab150077, Abcam).

Techniques: Expressing, Over Expression, Knockdown

Journal: The FASEB Journal

Article Title: Serpin family E member 1 enhances myometrium contractility by increasing ATP production during labor

doi: 10.1096/fj.202301804r

Figure Lengend Snippet: FIGURE 8 SERPINE1was directly activated by HIF-1α. (A) Genome browser view of HIF-1α and H3K27ac ChIP-seq signals across the SERPINE1 locus in hMSMCs under hypoxia. (B, C) The mRNA (B) and protein (C) expression levels of SERPINE1 in hMSMCs transferred with si-HIF-1α under hypoxia. (D) The protein expression levels of SERPINE1 in hMSMCs transferred with si-SERPINE1, SERPINE1 overexpression lentiviral particles, or si-HIF-1α under hypoxia. (E, F) The protein levels of SERPINE1 and HIF-1α in human in-labor myometrium were analyzed by western blotting (E) and their correlation analyzed (F). (G, H) The protein levels of SERPINE1 and HIF-1α in human non-labor myometrium were analyzed by western blotting (G) and their correlation analyzed (H). The bands of SERPINE1 and β-Actin were the same as those in Figure 6G,I. Data are expressed as mean ± SD, *p < .05, **p < .01.

Article Snippet: After blocking with 10% goat serum at 37°C for 1 h, the sections were stained at 4°C overnight with either rabbit polyclonal SERPINE1 antibody (1:200, NBP1- 19773, NOVUS), mouse polyclonal α- SMA (1:300, ab7817, Abcam), rabbit polyclonal ATP5PF antibody (1:1000, A3751, ABclonal), rabbit monoclonal SERPINE1 antibody (1:30, ab222754, Abcam), mouse monoclonal TOMM20 (1:100, ab7817, Abcam) followed by Alexa Fluor 647 Goat anti- mouse secondary antibody (1:500, ab15011, Abcam) or goat anti- rabbit Alexa Fluor 488- IgG (1:500, ab150077, Abcam).

Techniques: ChIP-sequencing, Expressing, Over Expression, Western Blot

Journal: The FASEB Journal

Article Title: Serpin family E member 1 enhances myometrium contractility by increasing ATP production during labor

doi: 10.1096/fj.202301804r

Figure Lengend Snippet: FIGURE 9 The effects of SERPINE1 on hMSMCs were rescued by HIF-1α. (A) The ATP levels of SERPINE1 in hMSMCs transferred with SERPINE1 overexpression lentiviral particles or si-HIF-1α under hypoxia. (B, C) OCR of hMSMCs transferred with SERPINE1 overexpression lentiviral particles or si-HIF-1α under hypoxia in response to oligomycin, FCCP and Rot/AA. (D, E) Gel contraction (D) and the gel areas (E) of hMSMCs SERPINE1 transferred with SERPINE1 overexpression lentiviral particles or si-HIF-1α under hypoxia. Data are expressed as mean ± SD, *p < .05, **p < .01. FCCP, fluorocarbonyl cyanide phenylhydrazone; OCR, oxygen consumption rate; Res., respiration; Rot/AA, rotenone/antimycin A.

Article Snippet: After blocking with 10% goat serum at 37°C for 1 h, the sections were stained at 4°C overnight with either rabbit polyclonal SERPINE1 antibody (1:200, NBP1- 19773, NOVUS), mouse polyclonal α- SMA (1:300, ab7817, Abcam), rabbit polyclonal ATP5PF antibody (1:1000, A3751, ABclonal), rabbit monoclonal SERPINE1 antibody (1:30, ab222754, Abcam), mouse monoclonal TOMM20 (1:100, ab7817, Abcam) followed by Alexa Fluor 647 Goat anti- mouse secondary antibody (1:500, ab15011, Abcam) or goat anti- rabbit Alexa Fluor 488- IgG (1:500, ab150077, Abcam).

Techniques: Over Expression