*2.1. Platelet-Rich Plasma (PRP) Preparation*

For the present experiments, thawed ready-to-use activated PRP aliquots classified as not suitable for transfusion-infusion purposes previously prepared and stored at −80 ◦C were used [48]. Briefly, PRP was collected from the blood of healthy adult donors subjected to plasma-platelet apheresis (Haemonetics MCS®, Haemonetics, Milan, Italy) as previously reported in detail [57]. The final platelet concentration in each PRP (without leukocytes) aliquot was 2 <sup>×</sup> 106 platelets/μL. Platelet activation was induced by the addition of a calcium digluconate solution (10%). The donors gave their written informed consent to allow the use of PRP for in vitro experimentations for which the Ethical Committee's approval is not required. PRP treatment was performed as previously reported [48,57].

#### *2.2. Cell Culture and Treatments*

Murine NIH/3T3 fibroblastic cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were grown in proliferation medium (PM), consisting of Dulbecco's Modified Eagle's Medium (DMEM; Sigma, Milan, Italy) containing 4.5 g/L glucose supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Sigma), at 37 ◦C in a humidified atmosphere of 5% CO2. Fibroblastic cells were induced to differentiate into myofibroblasts by shifting them in differentiation medium (DM) consisting of DMEM supplemented with 2% FBS and 2 ng/mL TGF-β1 (PeproTech, Inc., Rocky Hill, NJ, USA) for 48 h and 72 h, as previously reported [48]. In parallel experiments, to estimate the influence of PRP on fibroblast-myofibroblast transition, PRP was added to DM (1:50) [48,57]. In some experiments, the cells were cultured in PM, DM, or DM + PRP in the presence of 1 mM heptanol (Sigma), a specific GJ blocker, to evaluate the involvement of GJs in myofibroblastic differentiation.

#### *2.3. Electrophysiological Records*

Cell pairs were analyzed by the dual whole-cell patch-clamp technique, as previously reported [59–61]. Both passive membrane properties and GJ functionality were investigated. To this aim, cells were plated on glass coverslips (50,000 cells on each glass coverslips) to be located in the recording chamber and continuously superfused at a rate of 1.8 ml/min by a Pump 33 (Harvard Apparatus) with a physiological bath solution containing (mM) 140 NaCl, 5.4 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 D-glucose, and 5 HEPES (pH set at 7.4 with NaOH). The patch electrodes, pulled from borosilicate glass (GC 150–15; Clark, Reading, UK), were filled with the following solution (mM): 130 KCl, 10 NaH2PO4, 0.2 CaCl2, 1 EGTA, 5 MgATP, and 10 HEPES (pH was set to 7.2 with KOH). When filled, the pipette resistance ranged between 1.5 to 3.0 MΩ. Experiments were achieved at room temperature (22 ◦C). The set up for electrophysiological measurements was as previously reported [61] and consisted of the Axopatch 200 B amplifier (Axon Instruments, Union City, CA), an analog-to-digital/digital-to-analog interface (Digidata 1200; Axon Instruments), and pClamp 6 software (Axon Instruments). Currents were low-pass filtered at 1 kHz with a Bessel filter. The passive membrane properties, membrane resistance (Rm), and membrane linear capacitance (Cm) were consistently estimated in voltage-clamp starting from a holding potential (HP) of -70 mV and applying a 10-mV positive and negative step pulse. In brief, Rm was calculated using the relation: Rm = (ΔV − ImRa)/Im, where ΔV is the command voltage step amplitude, Im is the steady-state membrane current, and Ra the access resistance [23,62]. Cm was calculated from Cm = ΔQ(Rm + Ra)/RmΔV, corrected according to a previous report [63]. To properly compare the currents recorded from different cells, their values were normalized to Cm, assuming that the specific Cm was constant at 1 μF/cm2. The ratio I/Cm was intended as current density (in pA/pF). The junction potential of the electrode was estimated before making the patch (about −10 mV) and then was subtracted from the recorded membrane potential. The resting membrane potential (RMP) was recorded in current-clamp mode with a stimulus waveform: I = 0 pA. The protocol of stimulation used to record the currents flowing through GJs in voltage clamp and the recording procedure have been previously reported [61,64,65]. In brief, cell 1 of the pair was stepped from a holding potential (HP) of 0 mV, using a bipolar 5 s pulse protocol starting at trans-junctional voltage Vj = ±10 mV and ongoing at 20 mV increments up to ± 150 mV. The transjunctional current flowing through GJs is indicated as Ij. Precisely, the amplitude of Ij determined at the peak was named Ij,inst (instantaneous transjunctional current), whereas that measured at the end of each pulse is indicated as Ij,ss (steady-state transjunctional current). These values were used to calculate the related gap junctional conductances, Gj,inst and Gj,ss, by the ratios: Gj,inst = Ij,inst/Vj and Gj,ss = Ij,ss/Vj, respectively. The mean values of Gj,ss were normalized to those of Gj,inst, plotted as a function of Vj and fitted, when possible, with a Boltzmann function using the equation: Gj = (Gmax − Gmin)/(1 + exp (−A(Vj − V0))) + Gmin. In a set of experiments, heptanol (1 mM) was acutely applied to the bath solution to block gap junctional currents.

#### *2.4. Silencing of Cx43 Expression by Short Interfering RNA*

To inhibit the expression of Cx43, the cells were cultured either in a 6-wells/plate or on sterile glass coverslips put on the bottom of a 6-wells/plate in PM till a confluence of 80% and then transfected with a mix of short interfering RNA duplexes (siRNA; Santa Cruz Biotechnology, Santa Cruz, CA) corresponding to 3 distinct regions of the DNA sequence of mouse Cx43 gene (NM\_010288): 5 CCCAACUGAACCUUAAGAA3 , 5 CCUCACCAAAUGAUUUCUA3 , and 5 CCUACCAGUUUCUUCAAGU3 and/or with a non-specific scrambled (SCR)-siRNA (Santa Cruz Biotechnology) used as control. The siRNA transfections were performed according to manufacturer's instructions (Santa Cruz Biotechnology) and as previously reported [59]. Briefly, the cells were transfected with Cx43-siRNA duplexes or SCR-siRNA (20 nM) for 24 h and then shifted in fresh PM for additional 5 h. Thereafter, the transfected cells were cultured in DM with the addition or not of PRP for 48 h before being processed for Western blotting or immunofluorescence analysis of Cx43 and α-sma.

#### *2.5. Reverse Transcription - Polymerase Chain Reaction (RT-PCR)*

Cellular expression levels of Cx43 were evaluated by RT-PCR, as previously reported [48]. Briefly, according to manufacturer's instructions, total RNA was extracted from the cells cultured in the different experimental conditions on the wells of 6-wells/plates, by using TRIzol Reagent (Invitrogen, Life Technologies, Grand Island, NY, USA). One μg of total extracted RNA was reverse transcribed and amplified by using SuperScript One-Step RT-PCR System (Invitrogen, Life Technologies). cDNA synthesis was performed at 55 ◦C for 30 min; the samples were pre-denatured at 94 ◦C for 2 min and then subjected to 40 cycles of PCR performed at 94 ◦C for 15 s, alternating with 55 ◦C for 30 s and 72 ◦C for 1 min; the final extension step was performed at 72 ◦C for 5 min. The mouse gene-specific primers used were as follow: Cx43 (X61576.1), forward 5 -AACAGTCTGCCTTTCGCTGT-3 and reverse 5 -ATCTTCACCTTGCCGTGTTC-3'; β-actin (NM\_007393), forward 5 -ACTGGGACGACATGGAGAAG-3 and reverse 5 -ACCAGAGGCATACAGGGACA-3 . β-actin mRNA was used as an internal standard. Blank controls, consisting of no template (water), were performed in each run. The amplified samples were electrophoresed on 1.8% agarose gel containing ethidium bromide staining, and the intensity of the related bands was quantified by densitometric analysis by using ImageJ 1.49v software (NIH, https://imagej.nih.gov/ij/). Each band intensity was normalized to the relative β-actin.

#### *2.6. Confocal Laser Scanning Microscopy*

Cells grown on sterile glass coverslips in the different experimental conditions were fixed with paraformaldehyde (PFA) 0.5% diluted in PBS for 10 min at room temperature. Fixed cells were washed and permeabilized with cold acetone for 3 min, incubated with a blocking solution containing 0.5% bovine serum albumin (BSA, Sigma) and 3% glycerol in PBS for 20 min and thereafter incubated overnight at 4 ◦C with the following antibodies: mouse monoclonal anti-α-sma (1:100; Abcam, Cambridge, UK), rabbit polyclonal anti-Cx43 (1:250; Chemicon, Temecula, CA, USA), rabbit polyclonal anti-type-1 collagen (1:50; Santa Cruz Biotechnology), or mouse monoclonal anti-Cx26 (1:50; Sigma). The immunoreactions were revealed by specific anti-mouse Alexa Fluor 488- or 568 conjugated IgG or anti-rabbit Alexa Fluor 488- conjugated IgG (1:200; Molecular Probes, Eugene, OR, USA). In some experiments the fixed cells were incubated with Alexa Fluor 488-conjugated wheat germ agglutinin (WGA, 1:100; Molecular Probes) for 10 min at room temperature, which binds glycoconjugates present on cell membranes, or counterstained with propidium iodide (PI, 1:30 for 30 s; Molecular Probes), to detect nuclei. Negative controls were carried out by replacing the primary antibodies with non-immune serum, while cross-reactivity of the secondary antibodies was evaluated in control experiments in which primary antibodies were omitted. The immunolabeled samples were washed and mounted with an antifade mounting medium (Biomeda Gel mount, Electron Microscopy Sciences,

Foster City, CA, USA) to allow the observation under a confocal Leica TCS SP5 microscope equipped with a HeNe/Ar laser source for fluorescence measurements and differential interference contrast (DIC) optics (Leica Microsystems, Mannheim, Germany). Observations were performed by means of a Leica Plan Apo 63×/1.43NA oil immersion objective. A series of optical sections (1024 × 1024 pixels each; pixel size 204.3 nm) 0.4 μm in thickness were taken throughout the depth of the cells preparations at intervals of 0.4 μm, and the images were projected onto a single 'extended focus' image. Densitometric analyses of the intensity of α-sma, type-1 collagen, Cx43 and Cx26 fluorescence signals were performed on digitized images using ImageJ 1.49v software (NIH, https://imagej.nih.gov/ij/) in 20 regions of interest (ROI) of 100 μm2 for each confocal stack (at least 10).

#### *2.7. Western Blotting*

Total proteins extracted from the cells in the different experimental conditions were quantified, as reported previously [48]. Forty μg of total proteins were subjected to electrophoresis on NuPAGE®4%–12% Bis-Tris Gel (Invitrogen, Life Technologies; 200 V, 40 min) and blotted onto polyvinylidene difluoride (PVDF) membranes (Invitrogen, Life Technologies; 30 V, 1 h). The membranes were incubated with mouse monoclonal anti-α-sma (1:1000; Abcam), rabbit polyclonal anti-Cx43 (1:2500; Chemicon), and mouse monoclonal anti-Cx26 (1:500; Sigma) overnight at 4 ◦C. Immunodetection was performed according to the Western Breeze®Chromogenic Western Blot Immunodetection Kit protocol (Invitrogen, Life Technologies). The same membranes were subjected to the immunodetection of the expression of α-tubulin (rabbit polyclonal anti α-tubulin, 1:1000; Merck, Milan, Italy), assumed as control invariant protein. Densitometric analysis of the bands was performed using ImageJ 1.49v software (NIH, https://imagej.nih.gov/ij/), and the values normalized to control.

#### *2.8. Statistical Analysis*

Data were expressed as means ± standard error of the mean (S.E.M.) as a result of at least 3 independent experiments performed in triplicate. A 95% confidence level was used, assuming a normal distribution of values. Unpaired Student's t-test was used to compare the means of two conditions for independent data, statistically. The one-way analysis of variance (ANOVA) for any single independent variable was used to compare the differences between more than 2 groups and was followed by Tukey HSD or Bonferroni's post hoc adjustment. In electrophysiological experiments, 'n' indicates the number of cells analyzed. Values of *p* < 0.05 were considered statistically significant. Calculations were performed using GraphPad Prism software program (GraphPad, San Diego, CA, USA) and Microsoft Office Excel 2013 (Microsoft Corporation, Redmond, WA, USA).

#### **3. Results**
