**3. Results**

### *3.1. Suppressive Effect of Gemini-Cur on HCT-116 Cells*

Based on our previous reports, gemini surfactant nanoparticles significantly increase the cellular uptake of curcumin and suppress the proliferation of HCT-116 cells through the induction of apoptosis. Accordingly, Gemini-Cur significantly increases the proportion of SubG1 cells and induces apoptosis in HCT-116 cells compared to the non-treated group. Our Hoechst staining also illustrated the morphological characteristics of membrane shrinkage and nuclear fragmentation of HCT-116 cells. Here, we further confirm that Gemini-Cur modulates the growth of HCT-116 cells compared to void curcumin with IC50 value of 51.50 for 24 h (Figure 1A). In accordance with our previous work, acridine orange/ethidium bromide staining revealed that Gemini-Cur instigates apoptosis in colorectal HCT-116 cells. As Figure 1B shows, there is no significant apoptosis in non-treated cells (control). In contrast, the nucleus in dead cells reveals a condensed and granular forms with green–light orange fluorescence.

**Figure 1.** Gemini-Cur affects the proliferation of HCT-116 cells. (**A**): Gemini-Cur suppresses HCT-116 cells proliferation in a time- and dose-dependent manner. (**B**): Acridine orange/ethidium bromide staining also illustrated cells with apoptotic characteristics including membrane shrinkage and nuclear fragmentation in different incubation times (24, 48 and 72 h, Magnification ×400). Gemini: gemini surfactant nanoparticles; Cur: curcumin; Gemini-Cur: gemini curcumin.

#### *3.2. Raw Data Statistics and Quality Assessment*

Three replicates of treated samples and corresponding controls were subjected to RNA sequencing. On average, more than 40 million reads per sample were recorded (Figure 2A). The proportion of bases with high quality (Q30) was more than 90, indicating that the quality of RNA sequencing was proper for further analysis. After quality control and the removal of adaptors, more than 8 million reads were produced for each sample (Figure 2B).

**Figure 2.** Distribution of gene counts. As shown in bar plot (**A**), more than 8 million reads are produced for each sample. According to density plot, raw read Counts (log2 (counts + 1)) are not non-normalized distributed (**B**). The density plot of normalized count (log2 (normalized counts)) per sample is shown in plot (**C**). C1-3: controls; T1-3: Treated cells with Gemini-Cur.
