## 3 Results and Discussion
### Derivative **2** Interfaces with Actin Polymerization and Depolymerization
The fluorescence continued to decline until the end of the experiment, where it reached the value of 8400 RFU. The exposure of the actin monomers to CB produced a curve with a lesser decrease than the LA curve in the first 5 min (about 12,000 RFU), then the curve increased, ending at about 13,300 RFU. The curve obtained after the exposure to the derivative **2** (10 \(\upmu\)M) showed, in the initial part, a similar fashion to that of LA. In particular, the curve reached a lower RFU than the LA curve in the first 5 min (10,000 RFU). Then, the two curves superpose from 11 to 30 min, and in the last part, the curve of derivative **2** increased, reaching a final RFU of about 13,500. Figure 5: Effects of derivative **2** on tubulin polymerization. The effect of the derivative **2** on in vitro tubulin polymerization was evaluated by recording the turbidity difference at 350 nm for 3600 s (see the experimental section for further details). Paclitaxel was used as a tubulin-stabilizing agent, while Vinblastine and Nocodazole were used as tubulin-destabilizing agents. The three reference molecules and the derivative **2** were tested at the concentration of 10 \(\upmu\)M. DMSO was used as a negative control. The graphics are representative of three separated tests; standard deviations (SDs) are shown. Figure 6: \(\beta\)-Actin immunofluorescence studies. MCF-7 and MDA-MB-231 breast cancer cells and 3T3-L1 cells were exposed to the derivative **2** (25 \(\mu\)M), RSV (25 \(\mu\)M), or with the vehicle (CTRL, DMSO) for 24 h. CTRL cells and RSV-treated cells showed a normal organization of the actin filaments within  
actin system; contrarily, both the breast cancer cells exposed to the derivative **2** exhibited a strong disorganization of the actin system. Cells were monitored using the inverted fluorescence microscope at 40\(\times\) magnification. (**A**) DAPI (\(\lambda_{\text{ex}}\)/\(\lambda_{\text{em}}\) = 350/460 nm); (**B**) \(\beta\)-actin (Alexa Fluor\({}^{\text{\textregistered}}\) 568 (\(\lambda_{\text{ex}}\)/\(\lambda_{\text{em}}\) = 644/665 nm); (**C**) overlay channel. Representative fields are shown. To assess whether the derivative **2** could act also on F-actin disassembly, we performed a depolymerization assay (Figure 7B). First, we allowed the actin polymerization for one hour, and then we added the derivative **2**, in the same experimental condition used for the polymerization assay, monitoring the reactions for another hour. LA was used as a positive control and CB or vehicle (DMSO) were used as negative controls. In negative controls, the curves maintained the same initial RFU value until the end of the experiment, indicating no F-actin depolymerization. Instead, the LA curve decreased until a value of 10,000 RFU in the first 5 min (initial value 19,000 RFU), indicating that the actin depolymerization was occurring. Then, after  
Figure 7: **(A)** In vitro actin polymerization assay and (**B**) in vitro actin depolymerization assay. For both the assays, derivative **2** was used at the concentration of 10 uM, DMSO was used as a negative control, while actin-targeting agents, LA and CB (5 uM), were used as reference molecules. The formation of the actin filaments was measured with a microplate reader recording the fluorescence (\(\lambda_{\text{ex},\text{em}}\): 365/410 nm) in kinetic mode for 1 h. The graphics are representative of three separated tests; standard deviations (SDs) are shown. a small but not significant increase, the curve slowly decreased until the final value of 8000 RFU. The derivative **2** resulted also able to induce the F-actin depolymerization, with a similar effectiveness to LA. Indeed, in the initial part, the derivative **2** curve is to that of LA, reaching an RFU of 10,000 after only 4/5 min and maintaining it until 30 min. In the final part, the curve slowly increased, touching 12,000 RFU at the end of the experiment.