*1.3. Gap Junction Intercellular Communication (GJIC)*

The gap junction (GJ) channels are dynamic membrane domains built of two docking hemichannels called connexons assembled in the plasma membranes of two adjacent cells. Each connexon is a hexameric structure consisting of six transmembrane proteins named connexins (Cxs) that may have different molecular weights [49] and form an aqueous pore. The opening of these channels allows the flow of ions and small molecules (less than 1000 MW molecular weight size fractions) such as sugars, amino acids, oxygen, as well as second messengers such as cAMP, inositol phosphates, and calcium directly from one cell to another. The type of molecules (second messengers) passing through GJs can be influenced by the Cx isoform composition of the GJ channels. When the Cx isoforms are of the same type within a hemichannel, the resulting structure is called homomeric, whereas it is called heteromeric if more than one Cx isoform is present. The GJ channels composed of two identical hemichannels are named homotypic, and those consisting of two different hemichannels are named heterotypic. These two types of GJ channels exhibit peculiar and different gating properties influencing their voltage sensitivity [50]. GJIC is proposed to play a role in regulating the fibroblasts transition towards myofibroblasts as well as to be involved in the functional coupling of myofibroblasts to coordinate their activity [18,51–54]; however, GJ channel functionality and Cx composition during the phenotypic progression of fibroblasts into the myofibroblasts have not been fully elucidated yet and deserve more attention.

In the present in vitro study, by combining morphological, biomolecular, biochemical, and electrophysiological analyses, we extended our previous researches further exploring the potential molecular targets of the inhibitory action of PRP on myofibroblast generation. In particular, we focused the attention on the GJIC. The experimental model to evaluate fibroblast to myofibroblast transition has been previously validated [23,24,48,55–57] and consists in the culture of the cells in low serum conditions in the presence of TGF-β1. The treatment with PRP was also conducted as previously reported [48,57,58].

Here, while confirming the anti-fibrotic potential of PRP we provide the first experimental evidence that voltage-dependent GJ functionality and the expression of Cx43, a typical Cx forming voltage-dependent connexons, are important mechanisms by which TGF-β1 endorses fibroblasts differentiation towards myofibroblasts, and that PRP treatment hampers this effect. Moreover, we also demonstrated the involvement of not-voltage dependent GJs and Cx26, a typical Cx type forming not (or at least, scarcely) voltage-dependent connexons.

#### **2. Materials and Methods**
