3.7.4. Western Blotting

Preparation of the samples and Western blotting were performed as described previously [41]. For the detection of PSA and β-actin expression, the anti-PSA/KLK3 (Cell Signaling, #5365, 1:1000) and anti-β-actin-HRP (Santa Cruz, sc-1616, 1:10,000) antibodies were used. Treatment time was 24 h.

#### 3.7.5. Glucose Uptake Assay

The examination of the effect of the compounds on glucose uptake was carried out using PC-3 cells and the Glucose Uptake Cell-Based Assay Kit (Cayman Chemicals, Ann Arbor, MI, USA) and normalized to the cell viability measured using the MTS test [40]. 12,000 cells per well were seeded in two 96-well plates in 100 μL of media per well, incubated overnight, and treated with the drugs in 100 μL of FBS-free and glucose-free RPMI media per well for 24 h. For glucose uptake measurements, 10 μL of the 2-NBDG solution in FBS-free and glucose-free RPMI media (glucose uptake measurements, final 2-NBDG concentration in the wells was 50 μg/mL) of the vehicle (for cell viability measurements) was added to each well. After 6 h of incubation, the cells were washed twice with PBS (200 μL/well). Next, for the evaluation of glucose uptake, 100 μL of PBS was added to each well. The fluorescence was measured using Infinite F200PRO reader (TECAN, Männedorf, Switzerland). For cell viability measurements, the 100 μL of culture media containing MTS reagent was added to each well, and cell viability was measured using an Infinite F200PRO reader according to the manufacture's protocol.
