*5.4. Cell Proliferation Assay*

A WST-8 Cell Counting Kit (Wako, Osaka, Japan) was used to assess the proliferation of cells grown in 96-well microplates. Prostate cancer cells were seeded in DMEM medium containing 10% fetal bovine serum (FBS) in 96-well plates (5 × 10<sup>3</sup> cells/well), and a WST-8 assay was performed as previously described [16].

### *5.5. Analysis of the Cytotoxic Effect of NCL1 in Combination with Chloroquine*

The effect of a drug combination was calculated according to the median effect principle. First, we constructed dose-response curves for the cytotoxic effects of NCL1 and chloroquine, both alone and in combination, in 22Rv1 cells using a WST-8 assay. The data were used to determine the 'combination index' (CI), using the equation: CI = (D)1/(Dx)1 + (D)2/(Dx)2, where (D)1 and (D)2 are the combinations doses that kill x% of cells, and (Dx)1 and (Dx)2 are the doses of each drug alone that kill x% of cells. If CI < 1, then synergism is indicated as previously described [50,51].
