*4.6. Annexin V FITC/PI Assay*

To further confirm apoptosis, annexin V/FITC assay (ApoFlowEx FITC Kit, Exbio, Czech RepubliC), was employed to detect the mode of death in MDA-MB-231 cells. Briefly, the cells were seeded on 6-well plates for 24 h. After 24 h, cisplatin (13 μM) and Gemini-Cur (20 μM) was added to different wells in both singular and combination forms. After 24 and 48 h, cells were collected, washed twice with PBS and suspended in 1oo μL binding buffer. Annexin V/FITC solution was added to the cells followed by addition of 10 μL PI. Stained cells were then detected with a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

### *4.7. Expression Studies by Real-Time PCR and Western Blotting*

#### 4.7.1. Real-Time PCR

Total RNA was extracted with a selfmade TRIzol reagent, BRIzol, and followed by DNaseI treatment to eliminate any DNA content. The integrity and purity of RNA were evaluated using Pico200 (Picodrop, Hinxton, UK) and agarose gel electrophoresis. Total RNA was reverse transcribed into cDNA by employing PrimeScript RT Reagent Kit (Takara Bio, Kusatsu City, Japan). Real-time polymerase chain reaction (real-time PCR) was used to quantify the expression of BAX, BCL-2 and beta-2 microglobin (*β2M)*. Table 1 shows the characteristics of primers used in PCR. The comparative CT (2−ΔΔCT) method was used to determine the relative gene expressions [24].


**Table 1.** Sequences of primers used in real-time PCR.
