Sirius l

For Caspase-3/7 activity assays, LNCaP and C4-2cells were treated by JS-K in time-dependent manner and Caspase-Glo 3/7 assay was performed in 96-well plates. Then, an equal volume of Caspase-Glo 3/7 reagent was added into each well, and the cells were incubated for 30 min at room temperature in the dark. The luminescence was measured by a luminometer (Berthold Sirius L, Germany).

To examine cell apoptosis after treatment, caspase-3/7 activity was analyzed by Caspase-Glo 3/7 assays in accordance with the manufacturer’s protocol. Briefly, cells were seeded into 96-well dishes (Corning) and exposed to JS-K, conditioned medium, or NaNO₂. Then, an equal volume of Caspase-Glo 3/7 reagent was added into each well, and the cells were incubated for 30 min at room temperature in the dark. The luminescence was measured by a luminometer (Berthold Sirius L, Germany).

ATP levels were measured using an ATP Assay Kit (Beyotime) according to the manufacturer’s instructions. Briefly, bladder cancer cells were treated with JS-K (1 μM, 2 μM and 5 μM) for 6 h and incubated in 200 μL lysis buffer at 4 °C. The supernatant was then collected by centrifuging at 12,000 × g for 10 min at 4 °C. Subsequently, 50 μL of supernatant was added to 100 μL ATP detection reagent, and firefly luciferase activity was detected using a luminometer (Berthold Sirius L, Germany).

Intracellular ATP levels were measured using the ApoSENSOR cell viability assay kit (BioVision) according to the manufacturer’s instructions. Briefly, cells were treated with DHM (10, 50 and 100 μM) for 48 h, then incubated with 100 μl nuclear releasing reagent for 5 min at room temperature with gentle shaking, followed by further incubation with 5 μl ATP monitoring enzyme. Detection was performed using a luminometer (Sirius L; Titertek-Berthold, Pforzheim, Germany).

For the analysis of rat Ubiquitin (UbC) promoter, a 417 bp fragment flanking the transcription start point was amplified from rat genomic DNA. This fragment is similar to those characterized for human and rat promoters [20 (link)]. Sequences of all constructs were verified by automated DNA sequencing. The Cignal FoxO Reporter (luc) kit contains a mixture of inducible FoxO-responsive firefly luciferase construct and constitutively expressing Renilla luciferase construct (40:1).
For gene reporter analysis, cells were used at 80–90% confluence. Transfection was performed using LipofectAMINE2000 as described above. The DNA mixture comprised the pGL3-UbC luciferase reporter and the reference plasmid pRL-TK (ratio 95:5) or the Cignal FoxO Reporter mixture.
L6 myotubes were incubated in presence or absence of 25 μM HMB for 48 h. Cells were then incubated in the presence or absence of 5 μM DEX for 24 h. Luciferase activity was determined using the Dual Luciferase method (Promega) in a luminometer (Sirius L, Berthold Technologies, Bad Wildbad, Germany) and results were standardized for Renilla luciferase activity. To allow comparison of the expression patterns, the data are expressed as relative changes in luciferase activity and were normalized to a value of 100%.