Fluorometric assay kit

Caspase-3 activity was analyzed using a fluorometric assay kit (Abcam,
Cambridge, MA, USA). Cells were plated at 1 × 10⁶ cells in
100-mm Petri dishes in 10 mL of media overnight. Roniciclib (25 nmol/L) or
vehicle was added for 24 h. Adherent cells (5 × 10⁵) were
collected, centrifuged and lysed using 50 μL of lysis buffer on ice for
10 min, and incubated with DEVD-AFC substrate and reaction buffer at 37°C
for 1.5 h. Caspase-3 activity was detected by spectrophotometry. Each condition
was performed in duplicate.
The ability of roniciclib to induce sub-G1 apoptotic cell accumulation
was studied using flow cytometry. Cells were plated at 2 × 10⁵cells (BHP7–13 and WRO82–1) and 4 × 10⁵ cells
(K1 and FTC-133) per well in 6-well plates in 2 mL media overnight. Roniciclib
(25 nmol/L) or vehicle was added and incubated for 24 h. Floating cells and
trypsinized adherent cells were collected, washed with PBS, fixed with cold 70%
ethanol and incubated with RNase A (100 μg/mL; Sigma) and propidium
iodide (5 μg/mL; Sigma) at 37°C for 15 min. Apoptotic sub-G1 cells
were identified by DNA content analysis of 1 × 10⁴ events for
each sample by flow cytometry (BD FACScalibur Flow Cytometer, BD Biosciences,
Billerica, MA, USA). Each condition was performed in triplicate.

Caspase 1 activity was assessed in cell and pancreatic tissue lysates using a fluorometric assay kit (abcam,Cambridge, MA) and normalized to untreated cells in vitro. Trypsin activity was assessed in tissue lysates using a fluorometric substrate Boc-Gln-Ala-Arg-MCA as previously described.^{3 (link)} Myeloperoxidase activity was assessed in tissue lysates by chlorination of the substrate 3′-(p-aminophenyl) fluorescein (APF) using a commercial assay kit (Life Technologies). Pancreatic values were normalized to total amylase content in pancreatic tissue and untreated control samples. All fluorometric measurements were made in a Synergy Microplate Reader.

Lysosomal cathepsin D (CTSD) enzymatic activities were measured using a fluorometric assay kit from Abcam (Cat # ab65302) according to the manufacturer’s protocol in a 96-well assay format. Briefly, after isolating the lysosomes from WT and Ppt1^−/− mouse brain, lysosomes were washed three times with water, then lysed by Ca⁺⁺-free CD lysis buffer. Lysosomal lysates were then incubated in CD reaction buffer and CD enzymatic activity was measured according to manufacturer’s protocol. For measurement of TPP1 enzymatic activity, reaction was performed in a mixture of 100 μM Ala-Ala-Phe-7-amido-4-methylcoumarin (SIGMA, A3401), 10 μM trans-epoxysuccinyl-l-leucylamidobutane (E-64; SIGMA, E3132) and 1 μM Pepstatin A (Sigma, P5318) in 100 mM acetate buffer (pH=4.0), with 0.1% TritonX-100. Lysosomal lysates were incubated at 37 °C for 30 min. After incubation, TPP1 enzyme activity was stopped by addition of 100µl of 0.5 M glycine-carbonate buffer (pH=9.7). Each enzyme activity was measured as fluorescence by Flex station 3 microplate reader (Ex355nm/Em460nm). All assays were performed in triplicate.