*3.6. RNA Extraction and Quantitative Real-Time PCR*

After 48 h of treatment with Autumn Royal or Egnatia GSEs (20, 50, and 80 μg/mL), total RNA was extracted from Caco2 and SW480 cells using the Qiagen RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. The control sample was represented by untreated cells. Samples were retro-transcribed and analyzed using real-time Polymerase Chain Reaction (PCR) for the evaluation of 15-LOX-1 and PPAR-γ expressions on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer's instructions. Table 3 shows gene-specific primer sets used (Bio-Rad Laboratories); β-actin gene was chosen as the reference gene. The ΔΔCt method was used for relative quantification by CFX Manager software 2.1 (Bio-Rad Laboratories).



#### *3.7. FE-SEM Investigation*

Caco2 and SW480 cells were seeded on 1 cm2 silicon chips (Ted Pella Inc., Redding, CA, USA) at a density of 104 cells per well in eight-well chamber slides and, at a sub-confluent density, untreated control cells (CTR) were fixed at T0. To observe cell morphological changes, cell lines were exposed to increasing concentrations of Autumn Royal and Egnatia GSEs (10, 20, 50, and 80 μg/mL) for 24 (T1) and 48 h (T2). Cell lines were treated as previously described [53]. Briefly, each experimental time had a CTR group to highlight the cellular morphological differences before and after each time GSE exposure. As regards fixation procedures, the cells were treated with 3% glutaraldehyde in PBS for 1 h at 4 ◦C and then incubated for 1 h at room temperature in 1% osmium tetroxide (OsO4). Samples were washed several times with 0.005 M of sodium cacodylate pH 7.2, and the dehydration

was completed by passing the samples in increasing concentrations of acetone, from 20% to 100%, for 10 min each step. After completing the drying, samples were coated with a thin Au film using a sputter coater (208HR High Resolution Sputter Coater, Ted Pella Inc.). The fixed cells were observed by using a Zeiss Sigma FE-SEM (Carl Zeiss, Oberkochen, Germany), equipped with an in-lens secondary electron detector. The samples deposited on silicon chips were fixed to stainless-steel sample holders by using double-sided carbon tape. A uniform Au metal coating of few nanometers was deposited on the samples placed on silicon chips by using a turbomolecular pumped SC7620 Mini Sputter/Glow Discharge System of Quorum Technologies (Quorum Technologies Ltd, Lewes, UK). The overall FE-SEM measurements on the samples were acquired at constant Extra-High Tension (EHT) value of 3 kV and at working distance (WD) ranging from 1.8 to 3 mm. The FE-SEM micrographs presented for each experiment were selected as representative of a series of images collected on each sample. Each experiment was performed in triplicate.
