2.1.2. Synthesis of Gemini-Cur nanoparticles

Gemini-Cur nanoparticles were prepared by a nanoprecipitation method previously reported by our lab [16]. Briefly, we added 6 mg of Cur and 100 mg of gemini surfactants to 3 mL of methanol. Then, the solution was diluted twice in PBS under gently stirring condition, and the methanol was evaporated by using a rotary evaporator. The remaining solution was passed through a 0.22 μM syringe filter to remove possible contaminations and stored at 4 ◦C until use.

#### 2.1.3. Gemini-Cur Treatments

We have previously reported the IC50 values for Gemini-Cur on HCT-116 cells. Furthermore, we demonstrated that Gemini-Cur modulates the cell cycle and induces apoptosis in HCT-116 cells compared to controls [14]. To further confirm cellular toxicity on the cells treated with Gemini-Cur, we used ethidium bromide/acridine orange (EB/AO) staining. Briefly, the cells were left untreated or treated with Gemini-Cur at an IC50 dose onto 6-well plates. After 24, 48 and 72 h, the cells were detached by trypsin (0.25%; Sigma-Aldrich, USA) and transferred to glass slides. Staining solution (1 μL) containing 100 μg/mL acridine orange and 100 μg/mL ethidium bromide (Sigma-Aldrich, USA) was added to a suspension of HCT-116 cells. The cells were visualized under fluorescence microscopy (RX50, LABEX, England), and representative photographs were taken for further qualitative analysis. Fluorouracil (5-FU) apoptotic images were also provided as positive control.

For RNA sequencing, the cells were seeded on 6-well plates for 24 h and subsequently treated with Gemini-Cur. After 24 h, the cells were processed for RNA sequencing.

### 2.1.4. RNA Extraction and Preparation

According to the protocol of TRIzol reagent (Thermo Fisher Scientific, USA), total RNA was extracted from treated and non-treated HCT-116 cells. A total of six samples including three controls and three treated cells were incorporated in the study. After validating the integrity and purity (NanoDropTM, ThermoFisher, USA), all RNAs were treated with RNase-free DNase I to remove any DNA contamination. Then, RNAs were transferred to GeneTegra-RNA tubes (GenTegra Co., Seoul, Korea), dried in a freezer dryer (Sartorius Co., Germany) and sent to Macrogen Co., for sequencing (Macrogen Co., Seoul, Korea).
