Collagen type 1

At days 3 and 7 post-infection, wounded tissues were excised from euthanized mice; the tissues were fixed in 10% formalin and embedded in paraffin. Four micrometer vertical sections were cut and then fixed to glass slides and subjected to Haematoxylin and Eosin (H&E), Gram, MPO, or collagen type I (Santa Cruz Biotechnology, Dallas, TX, USA) mAb staining to assess morphology, bacterial burden, neutrophil infiltration, or collagen deposition, respectively. The slides were visualized using an Axiovert 40CFL inverted microscope (Carl Zeiss, Thornwood, NY, USA), and images were captured with an AxioCam MrC digital camera using the Zen 2011 digital imaging software. Quantification of the collagen staining was carried out using ImageJ software using threshold filters to isolate the stain and measurement of colorimetric intensity.

The rat PC12 cells were kindly provided by the Life and Health Sciences Research Institute (ICVS) of University of Minho. Cells were cultured in an expansion medium composed of DMEM medium (Sigma, Darmstadt, Germany) supplemented with 5% horse serum (HS; Sigma, Darmstadt, Germany), 10% fetal bovine serum (FBS; Life Technologies), and 1% antibiotic/antimycotic solution (ATB; Life Technologies) in a 75 cm² cell culture flask coated with 50 μg/mL collagen type I from rat tail (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Cells were cultured at 37 °C in a humidified incubator with 5% CO₂, and the medium was exchanged twice a week. For the assays, sub-confluent cells were harvested with TrypLE Express (1X) (Invitrogen, Bleiswijk, The Netherlands), seeded at a density of 20,000/eFM, and cultured in basal medium (BM: DMEM medium supplemented with 0.75% HS, 0.75% FBS, and 1% ATB). For the differentiation studies, PC12 cultured on eFM in BM supplemented with or without 100 ng/mL rat recombinant NGF-β (Sigma, Darmstadt, Germany) were carried out as positive or negative controls, respectively (Table 2). Cultures were incubated in a humid atmosphere at 37 °C and 5% CO₂, and retrieved for further analysis at predefined culturing times, namely 1, 3, and 7 days. All tests were carried out in triplicate and repeated at least three times (n = 3), independently.

One drop of each sample of soluble protein was placed in a nitrocellulose membrane. After drying, membranes were incubated for 1 h with 5% BSA, with agitation, at room temperature. Subsequently, membranes were incubated with primary antibody:mouse antiCollagen type I 1:1000 (#ab90395, abcam, Cambridge, UK); rabbit antiCollagen type IV, 1:500 (#ab6311, abcam, Cambridge, UK); rabbit antifibronectin 1:500 (#ab45688, abcam, Cambridge, UK); and mouse antilaminin, 1:500 (#L8271, Sigma-Aldrich, St. Louis, MO, USA). After overnight incubation, membranes were washed 3× for 5 min with Tris Buffered Saline (TBS) with Tween 20 and then the R.T.U. VECTASTAIN^® Universal ABC Elite^® Kit (#PK-7200, Vector Laboratories, Burlingame, CA, USA) was used as a secondary antibody, in accordance with manufacturer’s instructions. Finally, incubation was revealed using a Peroxidase Substrate Kit (DAB) (#SK-4100, Vector Laboratories, Burlingame, CA, USA). Extraction buffer without samples was used as a negative control. Collagen type I (#sc-136157, Santa Cruz Biotechnology, Santa Cruz, CA, USA), Collagen type IV (#C5533, Sigma-Aldrich), fibronectin (#FC010, Sigma-Aldrich, St. Louis, MO, USA) and laminin (#L6274, Sigma-Aldrich, St. Louis, MO, USA) were used as positive controls. At least three independent samples were used in each condition.

Nasal fibroblast lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto NC membranes (Bio-Rad, Berkeley, CA, USA). Membranes were blocked with 5% skim milk solution and they were incubated with antibodies against MMP-9 (Cell signaling, Beverly, MA, USA), collagen type I, fibronectin, TIMP-1, phosphorylated Smad (pSmad) 2/3, p38 mitogen-activated protein kinase (MAPK), and GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After incubation for 1 h, the membranes were washed and then treated with peroxidase-conjugated anti-rabbit immunoglobulin G (Santa Cruz Biotechnology). Bands were visualized using horseradish peroxidase conjugated secondary antibodies and an ECL system (Pierce, Rockford, IL, USA). The band densities were measured using the multi Gauge v.2.02 software (Fujifilm, Tokyo, Japan). The band intensities were expressed as a percentage of treated cells versus untreated cells.

Proteins of LV samples or cardiomyocytes were extracted by homogenizing samples in lysis buffer. Sample was loaded on SDS-PAGE gels and was then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). After being blocked with 5% bovine serum albumin (BSA), membranes were incubated with primary antibodies against AMPKα (Cell Signaling Technology, MA, USA), p-AMPK (Thr172) (Cell Signaling Technology, MA, USA), collagen type I (Santa Cruz Biotechnology, TX, USA), collagen type III (Santa Cruz Biotechnology, TX, USA), Lox (abcam, Cambridge, US), and MnSOD (abcam, Cambridge, US) overnight at 4 °C. Membranes were washed by TBST and further incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (Abbkine, CA, USA) at 37 °C for 2 h. Finally, protein blots were visualized by ECL Plus (Thermo Fisher Scientific, MA, USA) and the relative expression levels were normalized to a loading control of GAPDH (Cell Signaling Technology, MA, USA).