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Assessments of mechanisms leading to increased sTM levels following stimulation with S. aureus proteins. HUVEC monolayers were stimulated for 22 h with supernatant of overnight cultures of clinically isolated strains of S. aureus (M37, P08) and α-toxin. sTM was assessed in the host cell culture supernatant of stimulated cells (A). Thrombomodulin gene (THBD) expression in HUVECs after stimulation was assessed by qPCR (B) and total TM levels were measured after cell lysis of stimulated cells (C). LDH and TM were measured in cell culture supernatants after stimulations, with and without addition of 1300 nM broad-spectrum <t>MMP</t> inhibitor <t>Marimastat</t> (MMPI) or 10,6 µM ADAM10 selective inhibitor GI254023X (A10I) (D-E). LDH release measurements indicate cytotoxicity. Quantification of sTM and LDH are given in corrected optical density values. Asterisks indicate statistically significant differences (* = p < 0.05, ** = p < 0.01, *** = p < 0.001) according to ANOVA followed by Fisher’s least significant difference (LSD) post hoc test (A, B, C) or student t-test (D, E). Colors indicate different stimuli and symbols indicate different biological replicates. Direct cleavage of 40 ng rTM by 100 ng rADAM10 (rA10) was evaluated by western blot after 2 h incubation at 37 °C, with or without MMPI or A10I (F). Cleavage products are indicated by arrows at approximately 60 and 40 kDa.
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Assessments of mechanisms leading to increased sTM levels following stimulation with S. aureus proteins. HUVEC monolayers were stimulated for 22 h with supernatant of overnight cultures of clinically isolated strains of S. aureus (M37, P08) and α-toxin. sTM was assessed in the host cell culture supernatant of stimulated cells (A). Thrombomodulin gene (THBD) expression in HUVECs after stimulation was assessed by qPCR (B) and total TM levels were measured after cell lysis of stimulated cells (C). LDH and TM were measured in cell culture supernatants after stimulations, with and without addition of 1300 nM broad-spectrum <t>MMP</t> inhibitor <t>Marimastat</t> (MMPI) or 10,6 µM ADAM10 selective inhibitor GI254023X (A10I) (D-E). LDH release measurements indicate cytotoxicity. Quantification of sTM and LDH are given in corrected optical density values. Asterisks indicate statistically significant differences (* = p < 0.05, ** = p < 0.01, *** = p < 0.001) according to ANOVA followed by Fisher’s least significant difference (LSD) post hoc test (A, B, C) or student t-test (D, E). Colors indicate different stimuli and symbols indicate different biological replicates. Direct cleavage of 40 ng rTM by 100 ng rADAM10 (rA10) was evaluated by western blot after 2 h incubation at 37 °C, with or without MMPI or A10I (F). Cleavage products are indicated by arrows at approximately 60 and 40 kDa.
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Assessments of mechanisms leading to increased sTM levels following stimulation with S. aureus proteins. HUVEC monolayers were stimulated for 22 h with supernatant of overnight cultures of clinically isolated strains of S. aureus (M37, P08) and α-toxin. sTM was assessed in the host cell culture supernatant of stimulated cells (A). Thrombomodulin gene (THBD) expression in HUVECs after stimulation was assessed by qPCR (B) and total TM levels were measured after cell lysis of stimulated cells (C). LDH and TM were measured in cell culture supernatants after stimulations, with and without addition of 1300 nM broad-spectrum <t>MMP</t> inhibitor <t>Marimastat</t> (MMPI) or 10,6 µM ADAM10 selective inhibitor GI254023X (A10I) (D-E). LDH release measurements indicate cytotoxicity. Quantification of sTM and LDH are given in corrected optical density values. Asterisks indicate statistically significant differences (* = p < 0.05, ** = p < 0.01, *** = p < 0.001) according to ANOVA followed by Fisher’s least significant difference (LSD) post hoc test (A, B, C) or student t-test (D, E). Colors indicate different stimuli and symbols indicate different biological replicates. Direct cleavage of 40 ng rTM by 100 ng rADAM10 (rA10) was evaluated by western blot after 2 h incubation at 37 °C, with or without MMPI or A10I (F). Cleavage products are indicated by arrows at approximately 60 and 40 kDa.
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Assessments of mechanisms leading to increased sTM levels following stimulation with S. aureus proteins. HUVEC monolayers were stimulated for 22 h with supernatant of overnight cultures of clinically isolated strains of S. aureus (M37, P08) and α-toxin. sTM was assessed in the host cell culture supernatant of stimulated cells (A). Thrombomodulin gene (THBD) expression in HUVECs after stimulation was assessed by qPCR (B) and total TM levels were measured after cell lysis of stimulated cells (C). LDH and TM were measured in cell culture supernatants after stimulations, with and without addition of 1300 nM broad-spectrum <t>MMP</t> inhibitor <t>Marimastat</t> (MMPI) or 10,6 µM ADAM10 selective inhibitor GI254023X (A10I) (D-E). LDH release measurements indicate cytotoxicity. Quantification of sTM and LDH are given in corrected optical density values. Asterisks indicate statistically significant differences (* = p < 0.05, ** = p < 0.01, *** = p < 0.001) according to ANOVA followed by Fisher’s least significant difference (LSD) post hoc test (A, B, C) or student t-test (D, E). Colors indicate different stimuli and symbols indicate different biological replicates. Direct cleavage of 40 ng rTM by 100 ng rADAM10 (rA10) was evaluated by western blot after 2 h incubation at 37 °C, with or without MMPI or A10I (F). Cleavage products are indicated by arrows at approximately 60 and 40 kDa.
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Assessments of mechanisms leading to increased sTM levels following stimulation with S. aureus proteins. HUVEC monolayers were stimulated for 22 h with supernatant of overnight cultures of clinically isolated strains of S. aureus (M37, P08) and α-toxin. sTM was assessed in the host cell culture supernatant of stimulated cells (A). Thrombomodulin gene (THBD) expression in HUVECs after stimulation was assessed by qPCR (B) and total TM levels were measured after cell lysis of stimulated cells (C). LDH and TM were measured in cell culture supernatants after stimulations, with and without addition of 1300 nM broad-spectrum <t>MMP</t> inhibitor <t>Marimastat</t> (MMPI) or 10,6 µM ADAM10 selective inhibitor GI254023X (A10I) (D-E). LDH release measurements indicate cytotoxicity. Quantification of sTM and LDH are given in corrected optical density values. Asterisks indicate statistically significant differences (* = p < 0.05, ** = p < 0.01, *** = p < 0.001) according to ANOVA followed by Fisher’s least significant difference (LSD) post hoc test (A, B, C) or student t-test (D, E). Colors indicate different stimuli and symbols indicate different biological replicates. Direct cleavage of 40 ng rTM by 100 ng rADAM10 (rA10) was evaluated by western blot after 2 h incubation at 37 °C, with or without MMPI or A10I (F). Cleavage products are indicated by arrows at approximately 60 and 40 kDa.
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Assessments of mechanisms leading to increased sTM levels following stimulation with S. aureus proteins. HUVEC monolayers were stimulated for 22 h with supernatant of overnight cultures of clinically isolated strains of S. aureus (M37, P08) and α-toxin. sTM was assessed in the host cell culture supernatant of stimulated cells (A). Thrombomodulin gene (THBD) expression in HUVECs after stimulation was assessed by qPCR (B) and total TM levels were measured after cell lysis of stimulated cells (C). LDH and TM were measured in cell culture supernatants after stimulations, with and without addition of 1300 nM broad-spectrum MMP inhibitor Marimastat (MMPI) or 10,6 µM ADAM10 selective inhibitor GI254023X (A10I) (D-E). LDH release measurements indicate cytotoxicity. Quantification of sTM and LDH are given in corrected optical density values. Asterisks indicate statistically significant differences (* = p < 0.05, ** = p < 0.01, *** = p < 0.001) according to ANOVA followed by Fisher’s least significant difference (LSD) post hoc test (A, B, C) or student t-test (D, E). Colors indicate different stimuli and symbols indicate different biological replicates. Direct cleavage of 40 ng rTM by 100 ng rADAM10 (rA10) was evaluated by western blot after 2 h incubation at 37 °C, with or without MMPI or A10I (F). Cleavage products are indicated by arrows at approximately 60 and 40 kDa.

Journal: Virulence

Article Title: Staphylococcus aureus toxins mediate endothelial Thrombomodulin release during severe invasive infections

doi: 10.1080/21505594.2025.2605767

Figure Lengend Snippet: Assessments of mechanisms leading to increased sTM levels following stimulation with S. aureus proteins. HUVEC monolayers were stimulated for 22 h with supernatant of overnight cultures of clinically isolated strains of S. aureus (M37, P08) and α-toxin. sTM was assessed in the host cell culture supernatant of stimulated cells (A). Thrombomodulin gene (THBD) expression in HUVECs after stimulation was assessed by qPCR (B) and total TM levels were measured after cell lysis of stimulated cells (C). LDH and TM were measured in cell culture supernatants after stimulations, with and without addition of 1300 nM broad-spectrum MMP inhibitor Marimastat (MMPI) or 10,6 µM ADAM10 selective inhibitor GI254023X (A10I) (D-E). LDH release measurements indicate cytotoxicity. Quantification of sTM and LDH are given in corrected optical density values. Asterisks indicate statistically significant differences (* = p < 0.05, ** = p < 0.01, *** = p < 0.001) according to ANOVA followed by Fisher’s least significant difference (LSD) post hoc test (A, B, C) or student t-test (D, E). Colors indicate different stimuli and symbols indicate different biological replicates. Direct cleavage of 40 ng rTM by 100 ng rADAM10 (rA10) was evaluated by western blot after 2 h incubation at 37 °C, with or without MMPI or A10I (F). Cleavage products are indicated by arrows at approximately 60 and 40 kDa.

Article Snippet: The experimental conditions included the following treatments: (i) negative control, (ii) S. aureus overnight culture supernatants from strains M37 or P08 (7,000 ng/mL protein), (iii) 50 ng/mL recombinant α-toxin (IBT Bioservices, 1401–002), and (iv) co-treatment with 1300 nM broad-spectrum MMP inhibitor Marimastat (Tocris Bioscience, 2631; CAS: 154039-60–8) or 10,6 μM ADAM10 inhibitor GI254023X (Sigma-Aldrich, SML0789) in combination with S. aureus supernatants or recombinant α-toxin.

Techniques: Isolation, Cell Culture, Expressing, Lysis, Western Blot, Incubation