elisa assay  (R&D Systems)

Journal: Aging and Disease

Article Title: Role of the Senescence-Associated Factor Dipeptidyl Peptidase 4 in the Pathogenesis of SARS-CoV-2 Infection

doi: 10.14336/AD.2023.0812

Figure Lengend Snippet: DPP4 is upregulated in lung cells after induction of cellular senescence . IMR-90 cells were induced to senescence, and (A) DDP4 mRNA expression (n=4) and (B) soluble DPP4 in the supernatants of senescent cells compared to quiescent cells (n=8) were measured. ( C ) DPP4 is expressed in IMR-90, as detected by immunofluorescence staining, after the induction of senescence with irradiation (IR-induced senescence). Left: green DPP4; middle: IgG2 kappa Isotype control; right: nucleus stained with DAPI. ( D-F ) Senescence factors p16 , p21 , and MMP3 were significantly upregulated, whereas (G) Lamin-B1 was downregulated in senescent cells compared to quiescent cells (n=4). ( H ) Based on single-cell sequencing, DPP4 expression was determined 12 h, 24 h, 36 h, 2 d, 4 d, 6 d, 8 d, 10 d, and 12 d after senescence induction with doxorubicin (n=3). ( I ) In small airway epithelial cells, senescence was induced by IR and a significant upregulation of DPP4 mRNA was detected, associated with (J) significant p16 , (K) non-significant (ns) p21 , (L) significant MMP3 , and (M) ns Lamin-B1 regulation in senescent cells compared to quiescent cells (n=3). ( N-P ) In murine model, young (11 weeks, n=8) and old (26-32 months, n=14) mice were used for bronchoalveolar lavage. ( N ) soluble DPP4 was significantly upregulated. ( O ) DPP4 of lung homogenization was detectable as upregulated in old mice using Western blotting. ( P ) DPP4 activity was significantly upregulated (11 weeks n=3, 26-32 months n=14). Statistical analysis was performed under the assumption of non-normal distribution with the Mann-Whitney U test (ns p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001).

Article Snippet: The supernatants were analyzed using the human DPPIV/CD26 ELISA kit (#EHDPP4, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions.

Techniques: Expressing, Immunofluorescence, Staining, Irradiation, Sequencing, Homogenization, Western Blot, Activity Assay, MANN-WHITNEY

Journal: Aging and Disease

Article Title: Role of the Senescence-Associated Factor Dipeptidyl Peptidase 4 in the Pathogenesis of SARS-CoV-2 Infection

doi: 10.14336/AD.2023.0812

Figure Lengend Snippet: Patients with a severe course of COVID-19 infection show higher expression of DPP4 compared to control . ( A ) Venn-diagram showing age-matched proteins measured by SomaLogic protein assay using plasma from patients with mild (n = 35), moderate (n = 10), and severe (n = 42) COVID-19, as well as from controls (n = 33), and compared with the SASP identified from senescent IMR-90 cells. Differentially expressed genes were calculated using the R package DEseq2. p values were corrected for multiple comparison using Bonferroni correction. We selected a threshold of FDR < 0.05 and abs(logFC) > 1 for significance. Dashed box indicates further analyzed proteins. ( B ) Heatmap of 52 proteins of age-matched COVID-19 plasma samples with the SASP proteins of irradiated fibroblasts. ( C ) SASP proteins were highly upregulated: Dipeptidylpeptidase-4 (DPP4), Growth Differentiation Factor 15 (GDF15), Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Theta (YWHAQ), Stanniocalcin 1 (STC1), Parkinsonism Associated Deglycase (PARK7), Matrix Metallopeptidase 1 (MMP1), Heat Shock Protein Family A (Hsp70) Member 8 (HSPA8), Heat Shock Protein Family A (Hsp70) Member 1A (HSPA1A), Glutathione S-Transferase Pi 1 (GSTP1), Fibronectin 1 (FN1), Cathepsin Z (CTSZ), and Cathepsin D (CTSD).

Article Snippet: The supernatants were analyzed using the human DPPIV/CD26 ELISA kit (#EHDPP4, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions.

Techniques: Infection, Expressing, Comparison, Irradiation, Activation Assay

Journal: Aging and Disease

Article Title: Role of the Senescence-Associated Factor Dipeptidyl Peptidase 4 in the Pathogenesis of SARS-CoV-2 Infection

doi: 10.14336/AD.2023.0812

Figure Lengend Snippet: DPP4 upregulation upon senescence induction with higher pseudotyped virus but not infectious particle upload . ( A ) IMR-90 and Wi-38 fibroblasts were cultured at low population doubling levels (proliferative, P) or treated with etoposide to induce cellular senescence (S). DNA damage induced senescence (DDIS) was compared to replicative senescence (RS). Western Blotting for ACE2 and TMPRRS2 showed no difference in proliferative vs. senescent IMR-90 cells, but an increased signal in senescent Wi-38 cells was observed. DPP4 was upregulated in DDIS and RS Wi-38 cells, and p21 was increased in all conditions. ( B ) Senescence Associated-β-Galactosidase assays showed increased signals for all conditions (n=3). ( C ) BrdU incorporation was downregulated in senescent cells compared to proliferating cells (n=3). ( D ) Uptake of SARS-CoV-2 S (Spike) protein in proliferating vs. senescence cells. His-tagged SARS-CoV-2 Spike showed uptake in senescent cells (red signal), and nuclei stained in blue (DAPI). ( E ) Pseudoviral particles with a significantly higher luciferase luminescence signal in DPP4 overexpressing cells compared to knockout cells (DPP4Δ) and wild-type IMR-90 cells (n=3). ( F ) Significantly higher RNA detectable in SARS-CoV-2-infected IMR-90 cells after 24 h in DPP4 overexpressing cells compared to DPP4Δ and wild-type IMR-90 cells (n=6). ( G ) no differences after 48 h p.i. (n=6). ( H ) Detection of infectious viral particles (3 different SARS-CoV-2 isolates) using standard plaque assay to determine the plaque forming units per ml (PFU) revealed no differences between DPP4 overexpressing cells compared to DPP4Δ and wild-type IMR-90 cells after 8 h and 24 h p.i. (n=3). ( I ) Immunofluorescence staining showed no uptake of SARS-CoV-2 (red DPP4, green spike protein of SARS-CoV-2, blue nucleus (DAPI)). Statistical analysis was performed under the assumption of non-normal distribution with the Mann-Whitney U test (ns p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001).

Article Snippet: The supernatants were analyzed using the human DPPIV/CD26 ELISA kit (#EHDPP4, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions.

Techniques: Virus, Cell Culture, Western Blot, BrdU Incorporation Assay, Staining, Luciferase, Knock-Out, Infection, Plaque Assay, Immunofluorescence, MANN-WHITNEY

Journal: Aging and Disease

Article Title: Role of the Senescence-Associated Factor Dipeptidyl Peptidase 4 in the Pathogenesis of SARS-CoV-2 Infection

doi: 10.14336/AD.2023.0812

Figure Lengend Snippet: Mass spectrometry analysis of IMR-90 cells with data-independent acquisition (DIA) on the Orbitrap Eclipse Tribrid platform . ( A ) Supervised clustering using partial least square-discriminant analysis (PLS-DA) for IMR-90 wild-type (WT) cells, IMR-90 DPP4 overexpressing (OE) cells, IMR-90 DPP4 knockout (KO) cells, from both senescent and quiescent conditions. Senescent WT and DPP4-OE clustered together, but separately from senescent DPP4-KO, with 1,347 quantifiable protein groups. ( B ) Senescent DPP4-KO compared to senescent WT cells, with 39 significantly upregulated proteins. Protein Q-value < 0.01 & |Log2(FC)| > 0.58. ( C ) Heatmap showing upregulation of 39 proteins in senescent DPP4-KO compared to senescent WT cells. ( D ) Senescent DPP4-OE compared to senescent WT, with seven significantly altered proteins (six upregulated and one downregulated), Q-value < 0.01 & |Log2(FC)| > 0.58. ( E ) Heatmap showing upregulation of six proteins (including DPP4) and downregulation of complement factor C4A in senescent DPP4-OE compared to senescent WT cells. ( F ) Senescent DPP4-KO compared to quiescent DPP4-KO, with 147 significantly altered proteins (all upregulated). Q-value < 0.01 & |Log2(FC)| > 0.58. ( G ) Heatmap showing upregulation of ten proteins in senescent DPP4-KO compared to quiescent DPP4-KO cells.

Article Snippet: The supernatants were analyzed using the human DPPIV/CD26 ELISA kit (#EHDPP4, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions.

Techniques: Mass Spectrometry, Knock-Out

Journal: Aging and Disease

Article Title: Role of the Senescence-Associated Factor Dipeptidyl Peptidase 4 in the Pathogenesis of SARS-CoV-2 Infection

doi: 10.14336/AD.2023.0812

Figure Lengend Snippet: DPP4 mediates epithelial barrier disruption . ( A ) Transepithelial electrical resistance (TEER) in non-senescent (NS) and senescent corneal epithelial cells (SnC) was measured upon pre- and post-treatment with vehicle (DMSO), DPP4 inhibitor, or recombinant DPP4. ( B ) TEER performed post administration of conditioned media from NS and SnC treated with vehicle (DMSO), DPP4 inhibitor, or recombinant DPP4. ( C ) TEER in the different populations of corneal epithelial cells. WT: wild-type, OE: overexpression, KO: knockout. ( D ) Immunostaining of ZO-1 in NS/SnC cultures treated with DPP4 inhibitor, recombinant DPP4, or vehicle (DMSO). Scale bar indicates 50 µm. Results were plotted as the mean and standard deviation from four independent experiments, n=5. Statistical analysis was performed under the assumption of non-normal distribution with the Mann-Whitney U test (ns p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001).

Article Snippet: The supernatants were analyzed using the human DPPIV/CD26 ELISA kit (#EHDPP4, Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions.

Techniques: Disruption, Recombinant, Over Expression, Knock-Out, Immunostaining, Standard Deviation, MANN-WHITNEY