Zfr afc

Cancer cell lines were seeded in T25 flasks and, 24 h later, were treated as indicated in Section 3. After the removal of the medium, an extraction buffer containing different concentrations of digitonin (Merck) was used to separate cytosolic and total cathepsins. When necessary, the concentration of digitonin was optimized for different cell types. Cells were incubated with ice-cold lysis buffer (CelLytic^TM MT Mammalian Tissue Lysis/Extraction Reagent) containing a protease and phosphatase inhibitors cocktail (Merck) for 15 min at 4 °C on a rocking platform. Cysteine (Cys) and aspartate (Asp) cathepsin activities were measured using the fluorescent substrates zFR-AFC (AFC = 7-Amino-4-trifluoromethylcoumarin) (excitation at 405 nm; emission at 510 nm) and MCA-GKPILFFRLK(Dnp)-DR-NH2 [MCA = (7-methoxycoumarin-4-yl)acetyl; Dnp = dinitrophenyl] (excitation at 320 nm; emission at 420 nm) (Enzo Biochem, New York, NY, USA), respectively. Pepstatin A (5 mg/mL) and Leupeptin (50 mg/mL) (Merck) were used to inhibit the activity of aspartyl peptidases and serine-cysteine proteases, respectively.

Caspase-3 and cathepsin-B/L activity were determined in soleus muscle homogenates using the substrates Ac-DEVD-AMC (Alexis Biochemicals) and z-FR-AFC (Enzo Life Sciences, NY, USA), respectively, whereas calpain and 20S proteasome activity were determined using Suc-LLVY-AMC (Enzo-Life Sciences) [3 (link)]. These fluorogenic substrates are weakly fluorescent but yield highly fluorescent products following proteolytic cleavage by their respective proteases. Fluorescence was measured using a SPECTRAmax Gemini XS microplate spectrofluorometer (Molecular Devices, Sunnyvale, CA) with excitation and emission wavelengths of 360 nm and 440 nm (caspase-3), 380 nm and 460 nm (calpain and 20S proteasome), or 400 nm and 505 nm (cathepsin), respectively. To distinguish between calpain and 20S proteasome activity, the specific inhibitors Z-Leu-Leu-CHO (calpain; Enzo-Life Sciences) and epoxomicin (20S proteasome; Cayman Chemical, MI, USA) were incubated and the difference in fluorescence from homogenates incubated with and without the respective inhibitors was measured. All proteolytic activities were normalized to total protein content and expressed as fluorescence intensity in arbitrary units per mg of protein.

Lysosomal membrane permeabilization was assessed by cathepsin B release into the cytosol following a published protocol^{53 (link)}. Briefly, 5 × 10⁴ THP-1 cells were seeded in 24-well plates and treated with activators for 30 min in OPTI-MEM. Plasma membranes were permeabilized with 12 μg mL⁻¹ digitonin (Sigma, D141) in digitonin extraction buffer (250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 0.5 mM Pefabloc, pH 7.5) on ice for 10 min and plasma and lysosomal membranes were permeabilized with 200 μg mL⁻¹ digitonin in digitonin extraction buffer on ice for 10 min. Cathepsin B activity from the extract was measured in 50 mM sodium acetate, 4 mM EDTA, 8 mM DTT, 0.5 mM Pefabloc (Roche, 11873601001), 50 μM zFR-AFC (Enzo, ALX260-129-M005) using Ex 360 nm/Em 528 nm at 30 °C for 20 min using a Synergy HTX multi-mode reader (BioTek) and change in fluorescence over time was used to calculate enzyme activity. Cathepsin B activity was normalized to the extent of plasma membrane permeabilization as assessed by LDH activity. Normalized cathepsin B activity between 12 μg mL⁻¹ digitonin and 200 μg mL⁻¹ digitonin was used to calculate lysosomal membrane permeabilization as percent of maximum cathepsin B activity.