*4.3. Cell Treatment*

Gingival fibroblasts were seeded at a density of 1.0 × 10<sup>5</sup> cells/ml into 9 cm<sup>2</sup> (3 mL) wells and then subjected to serum starvation for 16 hours at 37 ◦C. Cells were treated with 1000 ng/mL amlodipine solution for 24 h. This solution was obtained in DMEM that was supplemented with 2% FBS, antibiotics and aminoacids. Cell medium alone was used as control negative. The cells were maintained in a humidified atmosphere of 5% CO2 at 37 ◦C. After the end of the exposure time, the cells were trypsinized and processed for RNA extraction.

### *4.4. RNA Isolation, Reverse Transcription and Quantitative Real-Time RT-PCR*

Total RNA was isolated from cultured cells using GenElute mammalian total RNA purification miniprep kit (Sigma-Aldrich, Inc., St Louis, Mo, USA), according to manufacturer's instructions. Pure RNA was quantified at NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, Massachusetts, USA).

cDNA synthesis was performed, starting from 500 ng of total RNA, using PrimeScript RT Master Mix (Takara Bio Inc, Kusatsu, Japan). The reaction was incubated at 37 ◦C for 15 min. and inactivated by heating at 70 ◦C for 10 s.

cDNA was amplified by Real Time Quantitative PCR using the ViiA™ 7 System (Applied Biosystems, Foster City, CA, USA).

All of the PCR reactions were performed in a 20 μL volume. Each reaction contained 10 μL of 2x qPCRBIO SYGreen Mix Lo-ROX (Pcrbiosystems, London, UK), 400 nM concentration of each primer, and cDNA. Table 2 reported the sequences of the primer that was used in the reaction.


**Table 2.** Primers sequences of SYBR® Green assay.

Custom primers belonging to the "Extracellular Matrix and Adhesion Molecules" pathway were purchased from Sigma Aldrich. All of the experiments were performed, including non-template controls to exclude reagents contamination. PCR was performed, including two analytical replicates.

The amplification profile was initiated by 10 min. incubation at 95 ◦C, followed by two-step amplification of 15 s at 95 ◦C and 60 s at 60 ◦C for 40 cycles. As final step, a melt curve dissociation analysis was performed.
