camostat  (BPS Bioscience)

Journal: Scientific Reports

Article Title: Dexamethasone enhances intestinal glucose absorption and TMPRSS2 expression with implications for hyperglycaemia and infection risk

doi: 10.1038/s41598-025-22312-8

Figure Lengend Snippet: Inflammation upregulates SGLT1, ACE2 and TMPRSS2 in Caco-2/TC7 cells. ( A ) Graphical illustration of the experimental protocol for the standard and inflamed Caco-2/TC7 cell models. Cells were differentiated in 25 mM glucose until day 7, then in 5.5 mM glucose until day 14. Cells were differentiated for 14 days from confluence and treated with dexamethasone (DEX) or 0.1% (v/v) DMSO (vehicle control) up to twice daily for 60 h, 24 h, or 4 h. For the inflamed model, a cytokine cocktail containing IL-1β (25 ng/mL) and TNF-α (50 ng/mL) was added to the basolateral (BL) once daily for 72 h. Finally, cell culture media from both apical (AP) and BL compartments were collected from the inflamed cells for IL-8 quantification. mRNA and protein were extracted from cells in both the standard and inflamed models and analysed through ddPCR and ELISA, respectively. A comparison of ( B ) IL-8 secretion and ( C ) SGLT1, ( D ) GLUT2, ( E ) ACE2 and ( F ) TMPRSS2 mRNA expression between standard (grey) and inflamed (blue) Caco-2/TC7 cells is shown. Total RNA was extracted and reverse transcribed, and absolute copies of SGLT1, GLUT2, ACE2 or TMPRSS2 cDNA measured by ddPCR are expressed relative to the housekeeping gene TBP. For IL-8 detection, cell culture media were processed, and IL-8 measured using ELISA and corrected for total protein measured by Bradford assay. Data are presented as mean ± SD (n = 6 technical replicates from N = 3 biological replicates). Differences between cells in the standard and inflamed environments in B-F were determined by unpaired t -test with Welch’s correction; ns—not significant, ** P < 0.01, *** P < 0.001. A —created with BioRender.com ( https://BioRender.com/r16s632 ); B-F —created with GraphPad Prism version 9.0.1.

Article Snippet: The human TMPRSS2 ELISA kit (NBP2-89171, manufactured by Novus Biologicals) was supplied by In Vitro Technologies (Noble Park North, VIC, Australia).

Techniques: Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Comparison, Expressing, Reverse Transcription, Bradford Assay

Journal: Scientific Reports

Article Title: Dexamethasone enhances intestinal glucose absorption and TMPRSS2 expression with implications for hyperglycaemia and infection risk

doi: 10.1038/s41598-025-22312-8

Figure Lengend Snippet: Gene expression changes in dexamethasone-treated and untreated normal and inflamed enterocytes from wild-type C57BL/6 J mice. ( A ) A summary of the GSE113691 study and the samples considered for analysis. Raw counts obtained from the NCBI GEO database ( GSE113691 ) were filtered (< 1 count per million in at least 3 samples) and were normalised using the trimmed mean of m-values (TMM). Differences in ( B ) SGLT1, ( C ) TMPRSS2, ( D ) GLUT2, ( E ) ACE2 gene expression in enterocytes from normal mice (NM) and inflamed mice (IF) between dexamethasone-treated (NM-D, IF-D) and untreated mice (NM-C, IF-C). Volcano plot showing ( F ) 910 differentially expressed genes between NM-D and NM-C and ( I ) 386 differentially expressed genes between IF-D and IF-C. Venn diagrams depicting upregulated and downregulated genes in ( G ) NM-D and ( J ) IF-D treatment groups compared to controls NM-C and IF-C, respectively. Red indicates upregulation and blue indicates downregulation (log 2 FC ≥ 1, FDR ≤ 0.05), overlapping indicates the unchanged number of genes. ( H ) Log 2 FC of SGLT1, TMPRSS2, GLUT2, and ACE2 between NM-D vs. NM-C and IF-D vs. IF-C (*indicates significantly differentially expressed genes (log 2 FC ≥ 1, FDR ≤ 0.05)). Gene Set Enrichment Analysis on the mouse Hallmark signature collection, showing positively and negatively enriched gene sets in ( K ) NM-D vs. NM-C and ( L ) IF-D vs. IF-C. Orange bars indicate gene sets enriched in both groups. ( M ) Pearson correlation matrix showing associations between SGLT1, TMPRSS2, GLUT2, and ACE2 mRNA expression in mouse intestinal epithelial cells. Blue represents positive correlation, red represents negative correlation. ( B-E ) Data are presented as mean ± SD (N = 3). ( B-E ) Significant differences were determined using one-way ANOVA with Fisher’s LSD multiple comparisons test. ns—not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. DEX—dexamethasone, NM-C—normal mouse control, NM-D—normal mouse treated with dexamethasone, IF-C—inflamed mouse control, IF-D—inflamed mouse treated with dexamethasone, NES—normalised enrichment score.

Article Snippet: The human TMPRSS2 ELISA kit (NBP2-89171, manufactured by Novus Biologicals) was supplied by In Vitro Technologies (Noble Park North, VIC, Australia).

Techniques: Gene Expression, Expressing, Control

Journal: Scientific Reports

Article Title: Dexamethasone enhances intestinal glucose absorption and TMPRSS2 expression with implications for hyperglycaemia and infection risk

doi: 10.1038/s41598-025-22312-8

Figure Lengend Snippet: Dexamethasone upregulates TMPRSS2 mRNA expression in Caco-2/TC7 cells. The acute and chronic effect of dexamethasone (DEX) on TMPRSS2 mRNA expression was assessed in 14-day differentiated Caco-2/TC7 cells, under either standard conditions (Panel 1) or an inflamed state induced by daily exposure to IL-1β and TNF-α during the final 72 h (Panel 2). Cells were treated with DEX (5, 10, 20 µM) or 0.1% (v/v) DMSO vehicle control for different time durations: ( A, D ) 4 h, ( B, E ) 24 h and ( C, F ) 60 h. A comparison of the effect of DEX between standard (grey) and inflamed (blue) conditions is shown in Panel 3 ( G —4 h, H —24 h, I —60 h). Total RNA was extracted and reverse transcribed, and absolute copies of TMPRSS2 cDNA measured by ddPCR are expressed relative to the housekeeping gene TBP. Values represent mean fold change compared to DMSO vehicle control (Panel 1) or cytokine-treated DMSO vehicle control (Panel 2). Data are presented as mean ± SD (n = 6 technical replicates from N = 3 biological replicates). Differences were determined as follows: A, D, G —unpaired t -test with Welch’s correction; B-C, E–F —one-way ANOVA with Fisher’s LSD multiple comparisons test; H, I —two-way ANOVA with Fisher’s LSD multiple comparisons test. ns—not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. Graphs created with GraphPad Prism version 9.0.1.

Article Snippet: The human TMPRSS2 ELISA kit (NBP2-89171, manufactured by Novus Biologicals) was supplied by In Vitro Technologies (Noble Park North, VIC, Australia).

Techniques: Expressing, Control, Comparison, Reverse Transcription

Journal: Scientific Reports

Article Title: Dexamethasone enhances intestinal glucose absorption and TMPRSS2 expression with implications for hyperglycaemia and infection risk

doi: 10.1038/s41598-025-22312-8

Figure Lengend Snippet: Pearson correlation analysis. ( A ) Pearson correlation matrix showing the association between variables. Blue represents a positive correlation, and red represents a negative correlation. ( B ) Table showing the statistical significance of the relationship between variables examined by Pearson correlation analysis. ACE2, TMPRSS2, SGLT1 and GLUT2 are mRNA expression levels; IL-8 is a secreted protein. The correlation matrix was created with GraphPad Prism version 9.0.1. ns—not significant; *** P < 0.001.

Article Snippet: The human TMPRSS2 ELISA kit (NBP2-89171, manufactured by Novus Biologicals) was supplied by In Vitro Technologies (Noble Park North, VIC, Australia).

Techniques: Expressing

Journal: The EMBO Journal

Article Title: Host cell and viral protease targets of human SERPINs identified by in silico docking

doi: 10.1038/s44318-025-00546-6

Figure Lengend Snippet: ( A ) Schematic of in-silico docking screen workflow. A panel of 48 respiratory proteases and 10 SERPINs upregulated upon viral infection were assembled. 3D structures were generated, with annotated protease active sites and SERPIN reactive center loops (RCLs). SERPIN-protease docking was performed using HADDOCK, guiding the RCL into the protease active site, and obtaining raw HADDOCK scores to reflect predicted binding energies. The dataset was normalized to the mean, and thresholds set for high-confidence in-silico binders (mean of positive controls) and high-confidence in-silico non-binders (mean of negative controls). Top-scoring complexes of interest were visually assessed for 3D RCL fit, exosite formation, and the positioning of protease active residues relative to SERPIN P4-P4’ residues. ( B ) Heatmap of docking results, with z-scores centered to the mean of control SERPIN-protease pairs and normalized for each SERPIN. The darker the red, the more favorable the binding energies. LEI leukocyte elastase inhibitor encoded by SERPINB1 , PAI-2 plasminogen activator inhibitor 2 encoded by SERPINB2 , Leupin encoded by SERPINB4 , PI-8 protease inhibitor 8 encoded by SERPINB8 , CAP-3 cytoplasmic anti-protease 3 encoded by SERPINB9 , Bomapin encoded by SERPINB10 , Headpin encoded by SERPINB13, ATIII antithrombin 3 encoded by SERPINC1, PAI-1 plasminogen activator inhibitor 1 encoded by SERPINE1, C1-INH C1 inhibitor encoded by SERPING1 . V validated pairs in vitro as shown in Fig. . ( C ) Normalized HADDOCK scores of PAI-1 with its positive controls (thrombin, active TMPRSS2, trypsin, kallikrein, uPA), negative controls (furin, uPA to PAI-1 P4-P4’ alanine substitutions, and uPA to PAI-1 P1 mutant R346A), the TMPRSS2 zymogen (TMPRSS2-z), and cathepsins. Green dotted line (mean of all positive controls in the in-silico screen) is the threshold for high-confidence binders; red dotted line (mean of all negative controls in the in-silico screen) is the threshold for high-confidence non-binders. Symbols: triangles, trypsin-like proteases; circles, subtilisin-like proteases; split circle, thrombin; hexagon, kallikreins; diamonds, cathepsins; light blue inactive serine protease, inactive; blue, serine proteases; yellow, cysteine proteases; salmon, aspartic proteases. Blue, serine proteases; yellow, cysteine proteases; red, aspartic proteases. ( D – F ) Docking structures of top-scoring complexes for PAI-1 with TMPRSS2 (zymogen), PAI-1 with TMPRSS2 (active) and CTSL, respectively. .

Article Snippet: TMPRSS2 Activity Assay Kit , BPS Bioscience , #78083.

Techniques: In Silico, Infection, Generated, Binding Assay, Control, Protease Inhibitor, In Vitro, Mutagenesis

Journal: The EMBO Journal

Article Title: Host cell and viral protease targets of human SERPINs identified by in silico docking

doi: 10.1038/s44318-025-00546-6

Figure Lengend Snippet: ( A , B ) Normalized reaction rates for uPA, TMPRSS2, cathepsin L, and cathepsin B in the absence or presence of PAI-1 ( A ), or PAI-2 ( B ) from in vitro fluorometric assays. Statistical analysis by One-way ANOVA and Kruskal–Wallis test, * P < 0.05. Exact P values in ( A ). uPA, 0 vs. 0.025 nM P = 0.3594, 0 vs. 0.25 nM P = 0.0146; TMPRSS2, 0 vs. 0.025 nM P = 0.3594, 0 vs. 0.25 nM P = 0.0146; CTSL, 0 vs. 0.025 nM P = 0.3594, 0 vs. 0.25 nM P = 0.0146; CTSB, 0 vs. 0.025 nM P > 0.9999, 0 vs. 0.25 nM P = 0.5934. Exact P values in ( B ). uPA, 0 vs. 1 nM P = 0.3594, 0 vs. 10 nM P = 0.0146; TMPRSS2, 0 vs. 1 nM P > 0.9999, 0 vs. 10 nM P = 0.9121; CTSL, 0 vs. 1 nM P > 0.9999, 0 vs. 10 nM P = 0.0722; CTSB, 0 vs. 1 nM P = 0.3594, 0 vs. 10 nM P = 0.0146. Mean ± SD, n = 3 replicates. Raw data in Dataset EV . ( C ) SDS-PAGE and silver stain of mixed recombinant active uPA (32 kDa, not visible) and PAI-1 (43 kDa). ( D ) SDS-PAGE and silver stain of mixed recombinant active TMPRSS2 (31 kDa) and PAI-1 (43 kDa). ( E ) SDS-PAGE and silver stain of mixed recombinant cathepsin L (32 kDa) and PAI-1 (43 kDa). *Denotes use of PAI-1 inhibitor triplaxinin and PAI-1 cleavage products. (F) SDS-PAGE and silver stain of mixed recombinant cathepsin L with PAI-1, reactions were conducted at pH 6.5 and pH 5.5. ( G ) SDS-PAGE and silver stain of mixed recombinant cathepsin B with PAI-1. ( H ) Schematic of importance of activity of TMPRSS2 and cathepsin L in SARS-CoV-2 entry by lineage. .

Article Snippet: TMPRSS2 Activity Assay Kit , BPS Bioscience , #78083.

Techniques: In Vitro, SDS Page, Silver Staining, Recombinant, Activity Assay

Journal: The EMBO Journal

Article Title: Host cell and viral protease targets of human SERPINs identified by in silico docking

doi: 10.1038/s44318-025-00546-6

Figure Lengend Snippet: ( A – C ) Schematic of fit of substrate residues into protease pockets for uPA, TMPRSS2, and cathepsin L, respectively. Substrates include zymogen self-cleavage region, canonical or previously described substrate, PAI-1 and fluorophore-linked peptide substrate used in this study. Amino acids are colored according to their side chain chemistry (Unipro UGENE): basic (R, K) litmus blue with R being more basic and darker; acidic (E, D) litmus red with more acidic being darker; hydrophobic (I, L, V, A), yellow with intensity corresponding to hydrophobic character; sulfur-containing (C, M) green; aromatic (F, Y, W) in teal; polar (N, Q, S, T) magenta/pink with darker coloring for more polarity; non-polar glycine (G) in dark gray and proline (P) in light gray. Assembled from references (Koga et al, ; Menard et al, ; Rossignol et al, ; Shrimp et al, ) and UNIPROT.

Article Snippet: TMPRSS2 Activity Assay Kit , BPS Bioscience , #78083.

Techniques:

Journal: The EMBO Journal

Article Title: Host cell and viral protease targets of human SERPINs identified by in silico docking

doi: 10.1038/s44318-025-00546-6

Figure Lengend Snippet: ( A – D ) SARS-CoV-2 WA-1 ( A , B ) or Omicron BA.1 ( C , D ) multi-cycle infection in Calu-3 cells with extracellular addition of buffer, 12.5 or 25 ng/mL of active PAI-1 or of 25 ng/mL heat-inactivated (HI) PAI-1 (48 hpi); or 10 or 100 ng/mL anti-PAI-1 depleting antibody or 10 ng/mL isotype IgG control (48 hpi) by high-content microscopy. Statistical analysis by One-way ANOVA and Holm-Sidak’s multiple comparison test, ** P < 0.005. Exact P values in ( A ). buffer vs. HI rPAI-1 P = 0.9407; buffer vs. rPAI-1 P < 0.0001; anti-PAI-1 (1:100) vs. IgG control P = 0.0059; anti-PAI-1 (1:50) vs. IgG control P < 0.0001. Exact P values in ( C ). buffer vs. HI rPAI-1 P = 0.5441; buffer vs. rPAI-1 P < 0.0001; IgG vs. anti-PAI-1 (1:100) P = 0.7968; IgG vs. anti-PAI-1 (1:50) P = 0.1473. n = 6 biological replicates. Representative images, DAPI (blue, nuclei), SARS-CoV-2 nucleoprotein (red). ( E ) Extracellular titers of SARS-CoV-2 WA-1 grown at low MOI-infection on Calu-3 cells for 48 h. Infectious titers were determined by TCID 50 assay on Calu-3. Statistical analysis by one-sided t test, * P < 0.05.Exact P value P = 0.05, n = 3 biological replicates ( F ). BHK cells co-transfected to express SARS-CoV-2 spike, TMPRSS2, and PAI-1. Spike S2 and 2’ band intensities by western blot and densitometry. % S2’ cleaved determined by ratio of S2’ vs S2’ + S2 band intensity from the blot shown. FL full-length. ( G ) BHK cells co-transfected to express SARS-CoV-2 spike and TMPRSS2, and rPAI-1 or buffer control added to the cell supernatant. Spike S2 and 2’ band intensities by western blot and densitometry. S2’ % cleaved determined by the ratio of S2 vs S2’ band intensity from the blot shown. FL full-length. .

Article Snippet: TMPRSS2 Activity Assay Kit , BPS Bioscience , #78083.

Techniques: Infection, Control, Microscopy, Comparison, Transfection, Western Blot