Journal: iScience

Article Title: Inhibition of cathepsin L ameliorates inflammation through the A20/NF-κB pathway in endotoxin-induced acute lung injury

doi: 10.1016/j.isci.2024.111024

Figure Lengend Snippet: Pharmacological inhibition of CTSL attenuates LPS-induced inflammatory response and ALI (A) BMDM were stimulated with LPS (100 ng/mL), or treated with SID26681509 (5 μM) for 6 h. Cells were then harvested to analyze secreted IL-6, CXCL1, and CXCL2 proteins in cell culture supernatants using ELISA, n = 3 each group. (B) HBE were stimulated with LPS (100 μg/mL) or treated with SID26681509 (1 μM) for 24 h. Cells were then harvested for analyzing secreted IL-6 and IL-8 proteins in cell culture supernatants using ELISA, n = 3 each group. (C) THP-1 were stimulated with LPS (100 ng/mL), or treated with SID26681509 (5 μM) for 6 h, Cells were then harvested for analyzing secreted IL-6, IL-8 proteins in cell culture supernatants using ELISA, n = 3 each group. (D) Mice were treated by intraperitoneal injection with SID26681509 (20 mg/kg) 1h before pentobarbital injection and then with LPS (in 50 μL saline) at a dose of 5 mg/kg or NS though intratracheal administration. Mice were sacrificed 24 h after LPS administration for analysis. Total and differential cell counts in the BALF from mice, n (DMSO) = 3, n (LPS) = 5, n (SID26681509) = 3, and n (SID26681509 + LPS) = 5. (E) Total protein concentration in the BALF. (F) Expression of IL6, CXCL1, and CXCL2 in the BALF. (G) Representative images of lung tissue with HE staining 24 h post-LPS challenge and histological inflammatory scores. Scale bar: 200μm. (H) For the survival assay, mice were treated intratracheally administered LPS (20 mg/kg) on day 0. Mice received an intraperitoneal injection of SID26681509 (20 mg/kg) or PBS 1 h before intratracheal instillation on day 0 and were injected with the drugs twice on day 3. Survival rates of mice after the intratracheal administration of LPS with or without IAAP intraperitoneal injection for 24 h. n (DMSO + LPS) = 17, n (SID26681509 + LPS) = 13. DMSO was used as the vehicle control. All the quantitative data are presented as mean ± SEM and differences were identified using one-way ANOVA. The log rank (Mantel-Cox) test was used to analyze survival rates. ∗ p < 0.05, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: SID26681509 , MedChemExpress , Cat#HY-103353.

Techniques: Inhibition, Cell Culture, Enzyme-linked Immunosorbent Assay, Injection, Saline, Protein Concentration, Expressing, Staining, Clonogenic Cell Survival Assay, Control

Journal: iScience

Article Title: Inhibition of cathepsin L ameliorates inflammation through the A20/NF-κB pathway in endotoxin-induced acute lung injury

doi: 10.1016/j.isci.2024.111024

Figure Lengend Snippet: CTSL regulates the LPS-induced K63-linked polyUb binding of NEMO by degrading A20 (A) BMDM cells were treated with IAAP or SID26681509 for 6 h, then stimulated with LPS (100 ng/mL) for 5 min, the levels of p -IKKα/β, IκBα, p -IκBα, p65, p-p65, and ACTB were examined using Western blotting. (B) BMDM cells were treated with IAAP or SID26681509 for 6 h, then stimulated with LPS (100 ng/mL) for 5 min. BMDM was analyzed for the spatial approximation of IKKγ with K63-linked polyUb components using PLA. Red, proximity ligation-positive signals. Scale bars: 5 μm. The quantification shown on the right represents the fluorescence, n = 15. (C) BMDM cells were treated with IAAP or SID26681509 for 6 h, then stimulated with LPS (100 ng/mL) for 5 min, the levels of A20, NEMO, IKKβ, and ACTB were examined using Western blotting. (D) BMDM cells were analyzed for the spatial approximation of A20 with CTSL components using PLA. Red, proximity ligation-positive signals. Scale bars: 5 μm. Quantification shown on the right represents the fluorescence, n = 15. (E) BMDM cells were transfected with control-, A20 -siRNA for 24 h. Then cells were treated with IAAP (5 μM) or SID26681509 (5 μM) for 6 h, and then stimulated with LPS (100 ng/mL) for 5 min. Cells were then harvested for analyzing the mRNA expression of IL-6 and CXCL1 using qRT-PCR, n = 3 in each group. DMSO was used as the vehicle control. All the quantitative data are presented as mean ± SEM and differences were identified using one-way ANOVA. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001; ns, p > 0.05.

Article Snippet: SID26681509 , MedChemExpress , Cat#HY-103353.

Techniques: Binding Assay, Western Blot, Ligation, Fluorescence, Transfection, Control, Expressing, Quantitative RT-PCR

Journal: iScience

Article Title: Inhibition of cathepsin L ameliorates inflammation through the A20/NF-κB pathway in endotoxin-induced acute lung injury

doi: 10.1016/j.isci.2024.111024

Figure Lengend Snippet:

Article Snippet: SID26681509 , MedChemExpress , Cat#HY-103353.

Techniques: Recombinant, Magnetic Beads, Transfection, BIA-KA, Activity Assay, In Situ, Enzyme-linked Immunosorbent Assay, Control, Software

Journal: Signal Transduction and Targeted Therapy

Article Title: Eosinophils promote pulmonary matrix destruction and emphysema via Cathepsin L

doi: 10.1038/s41392-023-01634-x

Figure Lengend Snippet: Cathepsin L derived from eosinophils are crucial for emphysema . a , b Mean linear intercepts were examined in mice lungs pretreated with 500 μg CTSL inhibitor (SID 26681509, quarterhydrate) or vehicle control 4 times 12 h before the first instillation of PPE. Scale bar: 200 μm. c , d Eosinophil counts and neutrophil counts were examined in mice lungs. e Mice were administered AAV-CTSL (5 × 10 12 vg/mL) in 60 μL PBS via intratracheal (i.t.) administration to cause CTSL expression in lungs 14 days before emphysema model establishment. Control mice were treated with controladeno-associated virus. The animals received intratracheal instillations of PPE or PBS weekly for 3 weeks on day 3 after the third challenge. After being sacrificed, CTSL expression in lung tissues of the control, control-si-CTSL, emphysema-si-NC, and emphysema-si-CTSL groups were examined. f Mean linear intercept in the treated mice lungs. Scale bar: 200 μm. g Cathepsin enzymatic activity was evaluated at 5-min intervals, specifically to monitor the generation of BODIPY FL-labeled fluorescent elastin fragments from self-quenching BODIPY FL-conjugated bovine neck ligament elastin. h Cathepsin activity was also assessed for the generation of fluorescein isothiocyanate (FITC) from FITC-labeled type I collagen. These data were obtained from three independent experiments and are highlighted as the mean ± s.e.m. * P < 0.05, ** P < 0.01 and *** P < 0.001 were analyzed using a two-tailed t- test. Panels a – f , n = 6 mice per group

Article Snippet: Inhibition experiments were carried out as previously described, with the addition of quarterhydrate (MCE, SID 26681509) before substrate introduction.

Techniques: Derivative Assay, Control, Expressing, Virus, Activity Assay, Labeling, Two Tailed Test