*4.2. Cell Viability Assay*

Cell viability was measured using the 3-[4,5-dimethylthiazol-2-yl] 2,5-iphenyltetrazolium bromide (MTT; Sigma-Aldrich, USA) assay. In brief, MDA-MB-231 cells were seeded into 96-well plates at a density of 10<sup>4</sup> cells per well and cultured overnight. After 24 h, cells were treated with various concentrations of cisplatin (0–200 μM), Gemini-Cur (0–100 μM) and combinations of Gemini-Cur and cisplatin (Gemini-Cur/Cis) for 24 and 48 h at 37 ◦C. Then, 20 μL of 5 mg/mL MTT was added to each well and further incubated for 4 h. The medium was replaced with 150 μL of DMSO and thoroughly mixed. Optical absorbance value was recorded at 570 nm using a plate reader (Biotek, ELX808, Winooski, VT, USA). The IC50 values of cisplatin, Gemini-Cur, and Gemini-Cur/Cis were calculated using the Graphpad prism software version 8.0.2. (Graphpad Prism, La Jolla, CA, USA).

#### *4.3. Analysis of Synergistic Cytotoxicity*

The synergistic effects of cisplatin and Gemini-Cur were quantitatively analyzed by the calculation of the combination index (CI) using Compusyn software program, which utilizes the Chou–Talalay equation method [23]. CI analysis calculates the interaction between Gemini-Cur and cisplatin agents. The CI value was calculated using the following equation:

$$\text{CI} = \frac{\text{CA.x}}{\text{ICx.A}} + \frac{\text{CB.x}}{\text{ICx.B}}$$

where CA.x and CB.x are the concentrations of agent A and agent B, respectively used in combination that inhibits cell growth by x%. The ICx.A and ICx.B are concentrations required for x% inhibition by agent A and agent B in singular form, respectively. Values of CI < 1, CI = 1, and CI > 1 indicate synergism, additive effect, and antagonism, respectively.

#### *4.4. Visualization of Apoptosis by Hoechst Staining*

MDA-MB-231 cells (4 × <sup>10</sup>5) were seeded into 6-well plates for 24 h. Then, the cells were treated with 13 μM cisplatin and 20 μM Gemini-Cur, both in singular and combination forms. After 48 h, treated cells were collected and fixed with 3.7% paraformaldehyde for 30 min at room temperature, washed and stained with 167 μmol/L Hoechst 33258 at 37 ◦C for 30 min. Finally, cells were observed under a fluorescence microscope (RX50, LABEX, London, England) equipped with a UV filter.

### *4.5. Cell Cycle Analysis by Flow Cytometry*

MDA-MB-231 cells (4 × 105) were seeded into 6-well plates and treated with 13 <sup>μ</sup><sup>M</sup> cisplatin and 20 μM Gemini-Cur in singular and combination forms for 24 and 48 h. Treated cells were fixed using ice cold 70% ethanol, washed 2X with PBS and then resuspended with propidium iodide (10 mg/mL) and ribonuclease A (0.1%) for 30 min. Finally, the cells were incubated for 30 min in the dark place at room temperature. Fluorescent events from propidium iodide–DNA complexes were quantified by fluorescence-activated cell sorter (FACS) (BD Biosciences, Franklin Lakes, NJ, USA) with a count of 10,000 cells per sample. Finally, DNA contents at different phases of the cell cycle were determined by using FlowJo 7.6.1 Software.
