In differentiated HL-60 cells, Merck AEBSF hydrochloride inhibited PMA-stimulated cytochrome-c reduction with a reported IC50 of 304 µM. This cellular readout does not establish selective inhibition of a purified NADPH oxidase. [2]
AEBSF can interfere with ROS detection: at 1 mM, the Merck material increased L012 chemiluminescence in a cell-free xanthine/XOD system without significantly reducing the paired cytochrome-c readout. L012 signals therefore require assay-interference controls. [2]
AEBSF hydrochloride as a trypsin-inhibition reference
Biochemical assay · Cell-free porcine pancreatic trypsin; BApNA substrate cleavage. [1]
AEBSF hydrochloride was the positive inhibitor control and had a reported trypsin IC50 of 0.003 mg/mL. This assay confirms protease-inhibitory activity; it does not establish covalent chemistry, irreversibility, or protease selectivity.
Literature-Reported Treatment Conditions
- Exposure duration
- 5 min preincubation at 37 °C, followed by 10 min substrate reaction at 37 °C (Section 2.5).
- Method
- Trypsin-inhibition assay in 20 mM Tris-HCl, pH 7.5; reported final enzyme concentration 60 µg/mL; BApNA substrate.
- Reported limits
- AEBSF concentration-series points and a distinct AEBSF stock vehicle are not tabulated in the selected original text. IC50 is an assay endpoint, not a treatment dose.
Methods and source locations
Compound reported in the paper: AEBSF hydrochloride; Compound label in the paper: AEBSF
Evidence for the named hydrochloride in the reported assay/model; no claim that the Adooq lot was tested.
Sections 2.1, 2.5, 2.8 and 3.2; Figure 2 and caption
Inhibition of trypsin-mediated BApNA cleavage: Absorbance at 410 nm after substrate cleavage; inhibition calculated relative to control absorbance. Assays in triplicate; paper reports results as mean ± SD, but does not supply an uncertainty for this AEBSF IC50. — Section 3.2 and Figure 2 (explicit AEBSF label); methods 2.5; statistics 2.8
AEBSF interference with an L012-based ROS readout
Biochemical assay · Cell-free xanthine/xanthine oxidase system, compared using L012 chemiluminescence and cytochrome-c reduction. [2]
At 1 mM, reported Merck AEBSF increased the L012 signal, while the cytochrome-c signal showed no significant suppression. The discrepancy demonstrates readout interference and limits interpretation of AEBSF experiments using L012.
Literature-Reported Treatment Conditions
- Treatment concentration
- 1 mM AEBSF (Figure 3 caption).
- Exposure duration
- 20 min equilibration before XOD addition; cytochrome-c readout at 10 min or L012 recording over 20 min.
- Method
- Xanthine/XOD assay with SOD and oxypurinol positive controls; untreated and solvent controls.
- Vehicle
- Hanks's balanced salt solution (HBSS).
- Reported limits
- Merck catalog number and batch are not specified; hydrochloride is defined in Abbreviations; no concentration or mass conversion is made.
Methods and source locations
Compound reported in the paper: AEBSF; Compound label in the paper: AEBSF
Evidence for the named hydrochloride in the reported assay/model; no claim that an Adooq product or lot was tested and no Adooq potency equivalence.
Methods: Xanthine/XOD activity assay, Materials, Data analysis; Results: Antioxidant effects; Figure 3A–B; Materials; Abbreviations glossary, AEBSF definition (PMC2970907.2)
L012 chemiluminescence in a NOX-independent superoxide-generating system: 100 µM L012; signals integrated over 20 min. Figure values are mean ± SEM, n ≥ 5 experiments. Exact fold change is not extracted. — Figure 3B and caption; Results: Antioxidant effects; Xanthine/XOD assay methods
Cytochrome-c reduction in the same xanthine/XOD assay comparison: 100 µM cytochrome c; absorbance at 540/550 nm, 10 min after reaction start. Xanthine-free blank subtracted. Figure values are mean ± SEM, n ≥ 5 experiments. — Figure 3A and caption; Xanthine/XOD assay methods
AEBSF attenuation of the HL-60 oxidative-burst readout
Cell-based study · DMSO-differentiated human HL-60 cells stimulated with PMA; cytochrome-c reduction readout. [2]
Reported AEBSF inhibited the PMA-stimulated cytochrome-c reduction signal with IC50 304 µM in the Figure 4 caption (rounded to 0.3 mM in prose). This is a cellular assay endpoint, not a purified-NOX potency or evidence of target selectivity.
Literature-Reported Treatment Conditions
- Treatment concentration
- Concentration-response series; 1 mM AEBSF used in the maximal-inhibition panel of Figure 4C. Individual series concentrations are not tabulated.
- Exposure duration
- 30 min inhibitor preincubation, then 120 min after PMA stimulation, at 37 °C.
- Method
- Cytochrome-c reduction in differentiated HL-60 cells. Signal specificity controls in Figure 2E include SOD, oxypurinol and L-NAME.
- Vehicle
- HBSS; solvent controls were performed for all assays and had no significant effect unless otherwise stated.
- Co-treatment
- PMA is the oxidative-burst stimulus, not a second therapeutic agent.
- Reported limits
- Merck catalog number and batch are not specified; hydrochloride is defined in Abbreviations. Methods describe duplicate assays repeated at least three times; Figure 4 explicitly gives n ≥ 5 experiments.
Methods and source locations
Compound reported in the paper: AEBSF; Compound label in the paper: AEBSF
Evidence for the named hydrochloride in the reported assay/model; no claim that an Adooq product or lot was tested and no Adooq potency equivalence.
Whole-cell NADPH oxidase activity assay; Materials; Data analysis; Results: Concentration dependence and efficacy; Figure 4C and caption; Figure 2E control; Materials; Abbreviations glossary, AEBSF definition (PMC2970907.2)
PMA-induced cytochrome-c reduction: 100 nM PMA and 100 µM cytochrome c; absorbance at 540/550 nm after 120 min at 37 °C. Blank subtracted and signal normalized to PMA-stimulated control. Figure values mean ± SEM, n ≥ 5 experiments; IC50-fit uncertainty not supplied. — Figure 4C/caption; Results: Concentration dependence and efficacy; Whole-cell assay methods
