Figure 2.
Figure 2. FSAP in serum is activated upon incubation with histones and protects against histone-induced cytotoxicity in HEK293 cells through histone proteolysis. (A) Serum samples (50%) of 3 healthy donors (HDs) were incubated with 100 µg/mL of calf thymus histones for 1 hour at 37°C. As a readout for subsequent activation (and inactivation) of FSAP in serum, complexes of FSAP with AP, a known serpin of FSAP, were determined by ELISA. (B-C) Histones (100 µg/mL) were incubated in the presence of different concentrations (0.6% to 5%) of HD serum or FSAP-deficient serum at 37°C for 30 minutes before their addition to HEK293 cells and overnight incubation. Lactate dehydrogenase (LDH) levels in the supernatant were quantified to determine cytotoxicity (B), whereas the viability of the cells was assessed by conversion of thiazolyl blue tetrazolium bromide (C). (D) FSAP was depleted from HD serum using an anti-FSAP antibody, and mock depletion is shown as a control. (E) Histones (100 µg/mL) were incubated with 0.6% to 5% FSAP-depleted (D) or mock-depleted serum, and the cytotoxicity for HEK293 cells was determined by LDH release. (F) Histone proteolysis after incubation in the presence of 1.3%, 2.5%, and 5% serum was determined in the samples from panel E by immunoblotting for histone H3. (G) Biotinylated histones (100 µg/mL) were incubated with 5% to 20% serum, mock-depleted serum, or FSAP-depleted serum, and histone proteolysis was assessed using streptavidin HRP. (H) Histones (100 µg/mL) were incubated with 50% HD serum or FSAP-depleted serum, and FSAP-AP complexes were determined at t = 0 to 30 minutes, whereas histone H3 degradation (inset) was assessed by immunoblotting for histone H3. FSAP-AP complexes were normalized to t = 30 minutes. (I) Histones (100 µg/mL) were incubated with 50% serum or plasma for 0 to 30 minutes, and histone H3 degradation was assessed by immunoblotting for histone H3. Data are expressed as mean ± standard error of the mean of 3 independent experiments, each performed in triplicate. *P < .05, **P < .01, ***P < .001 (calculated using an unpaired 2-tailed Student t test). def., deficient; depl., depleted.

FSAP in serum is activated upon incubation with histones and protects against histone-induced cytotoxicity in HEK293 cells through histone proteolysis. (A) Serum samples (50%) of 3 healthy donors (HDs) were incubated with 100 µg/mL of calf thymus histones for 1 hour at 37°C. As a readout for subsequent activation (and inactivation) of FSAP in serum, complexes of FSAP with AP, a known serpin of FSAP, were determined by ELISA. (B-C) Histones (100 µg/mL) were incubated in the presence of different concentrations (0.6% to 5%) of HD serum or FSAP-deficient serum at 37°C for 30 minutes before their addition to HEK293 cells and overnight incubation. Lactate dehydrogenase (LDH) levels in the supernatant were quantified to determine cytotoxicity (B), whereas the viability of the cells was assessed by conversion of thiazolyl blue tetrazolium bromide (C). (D) FSAP was depleted from HD serum using an anti-FSAP antibody, and mock depletion is shown as a control. (E) Histones (100 µg/mL) were incubated with 0.6% to 5% FSAP-depleted (D) or mock-depleted serum, and the cytotoxicity for HEK293 cells was determined by LDH release. (F) Histone proteolysis after incubation in the presence of 1.3%, 2.5%, and 5% serum was determined in the samples from panel E by immunoblotting for histone H3. (G) Biotinylated histones (100 µg/mL) were incubated with 5% to 20% serum, mock-depleted serum, or FSAP-depleted serum, and histone proteolysis was assessed using streptavidin HRP. (H) Histones (100 µg/mL) were incubated with 50% HD serum or FSAP-depleted serum, and FSAP-AP complexes were determined at t = 0 to 30 minutes, whereas histone H3 degradation (inset) was assessed by immunoblotting for histone H3. FSAP-AP complexes were normalized to t = 30 minutes. (I) Histones (100 µg/mL) were incubated with 50% serum or plasma for 0 to 30 minutes, and histone H3 degradation was assessed by immunoblotting for histone H3. Data are expressed as mean ± standard error of the mean of 3 independent experiments, each performed in triplicate. *P < .05, **P < .01, ***P < .001 (calculated using an unpaired 2-tailed Student t test). def., deficient; depl., depleted.

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