Fluorimetric assay

Treated and untreated cells were harvested 72 h after the treatment with different concentration of DOX or BNS-DOX, washed with 1× PBS, and subsequently resuspended in Annexin-V binding buffer (Life Technologies, Carlsbad, CA, USA) supplemented with 1:100 APC-conjugated Annexin-V (Immunotools, Friesoythe, Germany). Cells were analyzed by a FACS Calibur cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The analysis of apoptosis was performed by measuring the caspase 3 activity in cell lysates using a fluorimetric assay (BioVision, Milpitas, CA, USA) following the manufacture’ instructions.

Feces from each mouse of DIO3 were resuspended in a normal saline solution and crushed with a spatula. Then, the same volume of chloroform/methanol solution (2:1) was added to proceed with the lipid extraction. The suspension was homogenized and then centrifuged 10 min at 1000g. To collect the lipid phase, the bottom of the tube was pierced and the lipid phase was collected in a pre-weighed glass vial. Solvent from the lipid phase was evaporated under a hood for 24h. Then, the vials containing the lipids which had been extracted from feces, were weighed again to obtain the lipid mass. They were further resuspended in cholesterol buffer 10X (600μL assay buffer/g of feces) (Cayman Chemical) by strong vortexing.
Total cholesterol concentration was quantified in lipid feces suspensions using a colorimetric assay (Cayman Chemical) according to the manufacturer’s instruction.
Triglycerides concentration was quantified in lipid feces suspensions using a colorimetric assay (Cayman Chemical) according to the manufacturer’s instruction.
Fatty acid concentration was quantified in lipid feces suspensions using a fluorimetric assay (Biovision) according to the manufacturer’s instruction.

HaCaT keratinocytes were treated with triterpenoids (20 μM) for the indicated time and the activity of cathepsins was evaluated by a fluorimetric assay (BioVision Inc.). Briefly, HaCaT keratinocytes were lysed in 50–100 μL of chilled lysis buffer. After 10 min incubation on ice, cytosolic fraction was separated from the rest of the cell debris by centrifugation at 10,000 g at 4°C for 5 min and supernatant was retained for analysis of cytosolic cathepsins related to LMP. Alternatively, whole-cell extracts were obtained after lysing the cell debris above with more 50–100 μL of chilled lysis buffer followed by freezing/thawing cycles (3×). Next, these whole-cell lysates were centrifuged at 10,000 g for 10 min at 4 °C, and 15 μg of protein per sample was used for enzymatic assays Protein concentration in the same aliquot was measured using the BRADFORD assay (Bio-Rad Laboratories, Hercules, CA, USA) and enzyme activity normalized by total protein and expressed as arbitrary units (a. u.) after ratio to control (DMSO). The quantification of lysosome cathepsin was performed by subtracting from the total cathepsin the cytosolic cathepsin activity.