*4.4. Data Analysis*

The viability dose–response curve of the compounds was analyzed using the Emax model (Equation (1)).

$$\% \text{ Cell viability} = (100 - \text{R}) \times \left(1 - \frac{[\text{D}]^m}{K\_d^m + [\text{D}]^m} \right) + \text{R} \tag{1}$$

where R is the residual unaffected fraction (the resistance fraction) which is deduced from fitting concentration versus viability on the *Emax* equation (Equation (1)) described above, [D] is the drug concentration used, *Kd* is the drug concentration that produces a 50% reduction in the maximum inhibition rate and *m* is a Hill-type coefficient. IC50 was defined as the drug concentration required to reduce optical density to 50% of that of the control (i.e., *Kd* = IC50 when R = 0 and *Emax* = 100 − R) [68].

### *4.5. Apoptosis*

Annexin V conjugates allow for the identification of cell surface changes that occur early during the apoptotic process using flow cytometry. Early in the apoptotic process, phosphatidylserine emerges from within the cytoplasmic membrane and becomes exposed on the cell surface, which is thought to be important for macrophage recognition of cells undergoing apoptosis. The binding of Annexin V to phosphatidylserine is calcium-dependent, reversible, and specific with a *Kd* of approximately 5 × 10 − 10 M [69].

#### *4.6. Assessment of Active Caspase-3 Concentration*

To assess the effect of GCB, terrein, and their combination on apoptosis, the active caspase-3 concentration was measured using a Quantikine® caspase-3 ELISA Kit (R&D Systems, Inc., Minneapolis, MN, USA). Briefly, the cells were exposed to the predetermined IC50s of test compounds (single or combined treatments) or drug-free media (control group) for 24 h. Cells were harvested and washed twice with PBS, then incubated with the biotin-ZVKD-fmk inhibitor for 1 h. Cells were transferred into the wells of a microplate precoated with a monoclonal antibody specific for caspase-3. Following a wash to remove any unbound substances, streptavidin conjugated to horseradish peroxidase was added to the wells and bound to the biotin on the inhibitor. Following a wash to remove any unbound streptavidin–HRP, a substrate solution was added to the wells. The enzyme reaction yields a blue product that turned yellow when a stop solution was added. The optical density of each well was determined within 30 min, using a microplate reader set to 450 nm with a wavelength correction at 540 nm or 570 nm. The concentrations of active caspase-3 were calculated from a standard curve constructed with known concentrations of active caspase-3. Caspase concentration was expressed as ng/mg protein. Proteins were determined by the Bradford method using purified bovine serum albumin as a standard protein.

#### *4.7. Autophagy*

Acridine orange (AO) is a cell-permeable green fluorophore that can become hydronated and consequently absorbed by acidic vesicular organelles. Its metachromatic shift from green to red fluorescence is highly dependent on its concentration, which causes AO to fluoresce from green to red in acidic organelles, such as lysosomes. Lysosomes tend to increase in number and volume when autophagy occurs; AO staining is a quick and reliable method for the assessment of autophagy [70].
