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SUPPLEMENTARY METHODS Morphological and Structural Analysis of Pkecm After Fixation with 2.5 % Glutaraldehyde Dehydration In

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SUPPLEMENTARY METHODS Morphological and Structural Analysis of Pkecm After Fixation with 2.5 % Glutaraldehyde Dehydration In Electronic Supplementary Material (ESI) for Biomaterials Science. This journal is © The Royal Society of Chemistry 2020 SUPPLEMENTARY METHODS Morphological and structural analysis of pKECM After fixation with 2.5 % glutaraldehyde dehydration in a graded ethanol series, pKECM structure was analyzed by Scanning Electron Microscopy (SEM; JSM-6010 LV, JEOL, Japan). Samples were sputter coated with gold (coater 108 A, Cressington, United States) prior to examination. Micrographs were obtained at an accelerating voltage of 10 kV. The infrared spectra of pKECM before and after sterilization was analyzed by Attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FITR, IRPrestige 21, Shimadzu), with a spectral range of 500-3000 cm-1 in reflection mode to characterize the composition and molecular conformation of the pKECM. Thermal properties of the pKECM substrates before and after sterilization were characterized by differential scanning calorimetry (DSC, Q100, TA Instruments). Approximately 2 mg of sterile and non-sterile pKECM were transferred into an aluminum pan and the sample was scanned with a modulated heating method. First, pKECM was heated until 250 C at a heating rate of 10 C min-1 and then cooled down to 10 C. The sample chamber was purged with nitrogen at a flow rate of 50 mL h-1. Histological and biochemical analysis of pKECM pKECM substrate was assessed by Hematoxylin & Eosin (H&E) and Masson’s Trichrome (MT) stainings of frozen sections. The samples were fixed for 30 min with 10 % neutral-buffered formalin before being embedded in OCT compound (Bio-optica). After this, they were snap-freezed in liquid nitrogen and cut into 10 m sections using a cryostat. Sections were stained with working solutions of H&E and a MT kit according to manufacturer’s instructions (Bio-optica), dehydrated through a series of ethanol and mounted. For the detection of dsDNA, pKECM was digested for 2 h at 56 C with proteinase K and the remaining DNA extraction was performed using DNeasy blood and tissue kit (Qiagen) according to manufacturer’s instructions. The digested samples were 1 quantified using Quanti-iT PicoGreen dsDNA assay kit (Invitrogen) according to the manufacturer’s protocol. Values were extrapolated using a standard curve of dsDNA and sample fluorescence was read in a microplate reader (excitation: 485/20 nm; emission: 530/25 nm). Characterization and relative quantification of protein composition by nano-liquid chromatography–tandem mass spectrometry (nanoLC- MS/MS) Each sample was reduced and alkylated and processed for proteomics analysis following the solid- phase-enhanced sample-preparation (SP3) protocol as described in PMID30464214.1 Enzymatic digestion was performed with Trypsin/LysC (2 g) overnight at 37 ºC at 1000 rpm. Protein identification and quantitation was performed by nanoLC-MS/MS. This equipment is composed by an Ultimate 3000 liquid chromatography system coupled to a Q-Exactive Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany). Samples were loaded onto a trapping cartridge (Acclaim PepMap C18 100Å, 5 mm x 300 µm i.d., 160454, Thermo Scientific) in a mobile phase of 2 % acetonitrile (ACN), 0.1% formic acid (FA) at 10 µL min-1. After 3 min loading, the trap column was switched in-line to a 50 cm by 75 μm inner diameter EASY-Spray column (ES803, PepMap RSLC, C18, 2 μm, Thermo Scientific, Bremen, Germany) at 250 nL min-1. Separation was generated by mixing A: 0.1% FA, and B: 80 % ACN, with the following gradient: 5 min (2.5 % B to 10 % B), 120 min (10 % B to 30 % B), 20 min (30 % B to 50 % B), 5 min (50 % B to 99 % B) and 10 min (hold 99 % B). Subsequently, the column was equilibrated with 2.5 % B for 17 min. Data acquisition was controlled by Xcalibur 4.0 and Tune 2.9 software (Thermo Scientific, Bremen, Germany). The mass spectrometer was operated in data-dependent (dd) positive acquisition mode alternating between a full scan (m/z 380-1580) and subsequent HCD MS/MS of the 10 most intense peaks from full scan (normalized collision energy of 27 %). ESI spray voltage was 1.9 kV. Global settings: use lock masses best (m/z 445.12003), lock mass injection Full MS, chrom. peak width (FWHM) 15 s. Full scan settings: 70 k resolution (m/z 200), AGC target 3E6, maximum injection 2 time 120 ms. dd settings: minimum AGC target 8E3, intensity threshold 7.3E4, charge exclusion: unassigned, 1, 8, > 8, peptide match preferred, exclude isotopes on, dynamic exclusion 45vs. MS2 settings: microscans 1, resolution 35 k (m/z 200), AGC target 2E5, maximum injection time 110 ms, isolation window 2.0 m/z, isolation offset 0.0 m/z, spectrum data type profile. The raw data was processed using Proteome Discoverer 2.4.0.305 software (Thermo Scientific) and searched against the UniProt database for the Sus scrofa Proteome 2019_11. The Sequest HT search engine was used to identify tryptic peptides. The ion mass tolerance was 10 ppm for precursor ions and 0.02 Da for fragment ions. Maximum allowed missing cleavage sites was set 2. Cysteine carbamidomethylation was defined as constant modification. Methionine oxidation and protein N- terminus acetylation were defined as variable modifications. Peptide confidence was set to high. The processing node Percolator was enabled with the following settings: maximum delta Cn 0.05; decoy database search target FDR 1 %, validation based on q-value. Protein label free quantitation was performed with the Minora feature detector node at the consensus step. Precursor ions quantification was performing at the processing step with the following parameters: unique plus razor peptides were considered for quantification, precursor abundance was based on intensity and normalization was based on total peptide amount. The total number of proteins identified in each fraction was obtained by filtering peptide identifications at > 95.0 % confidence interval with at least two peptides per protein. Gene ontology analysis (GO) was performed manually using AmiGO online platform.2,3 Following categorization, non-ECM proteins were excluded to perform graphical analysis. Coefficient of variation (CV) values were calculated by dividing standard deviation by mean values of the ECM proteins identified on each kidney sample. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE 4 partner repository with the dataset identifier PXD019122. Isolation and characterization of human renal progenitor cells (hRPC) 3 Human kidney fragments were obtained in agreement with the Ethical Committee on human experimentation of the Azienda Ospedaliero-Universitaria Careggi, Florence, Italy. All subjects gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Ethics Committee on Human Experimentation of the Azienda Ospedaliero-Universitaria Careggi, Florence, Italy (project identification code 2015/0009082 from 25/03/2015). Human RPCs were obtained and cultured as previously described,5 with minor modifications. Briefly, we minced the cortex and we performed an enzymatic digestion with collagenase solution type IV 750 U mL-1 (Sigma Aldrich) in PBS for 45 min at 37 °C. After digestion, we filtered the solution by a standard sieving technique through graded mesh screens (60 and 80 mesh). Subsequently, we performed red blood cell lysis by incubating with lysis buffer (NH4Cl 0.087 %) for 4 min at 37 °C. The cellular suspension was collected, washed and plated on petri dish containing complete medium. The medium is composed by Microvascular Endothelial Cell Growth Medium (EGM™-MV, Lonza) supplemented with 20 % HyClone™ Fetal Bovine Serum (FBS, GE Healthcare). After 4 to 5 days of culture, cells that adhered to the plate were subcultured. Cultures were checked for simultaneous expression of CD133 and CD24 by flow cytometry, according to previously published methods for characterizing hRPC.6 For the analysis, total hRPCs population was gated on a forward scatter (FSC)/side scatter (SSC) plot and dead cells were excluded by Propidium Iodide (PI) staining. Live cells (PI negative) were gated for CD133 and CD24 expression. Only cells between passage 1-3 and expressing > 90 % CD24/CD133 double positivity were used in this study. 4 REFERENCES 1 C. S. Hughes, S. Moggridge, T. Müller, P. H. Sorensen, G. B. Morin and J. Krijgsveld, Nat. Protoc., , DOI:10.1038/s41596-018-0082-x. 2 S. Carbon, A. Ireland, C. J. Mungall, S. Shu, B. Marshall, S. Lewis, J. Lomax, C. Mungall, B. Hitz, R. Balakrishnan, M. Dolan, V. Wood, E. Hong and P. Gaudet, Bioinformatics, , DOI:10.1093/bioinformatics/btn615. 3 S. Carbon, E. Douglass, N. Dunn, B. Good, N. L. Harris, S. E. Lewis, C. J. Mungall, S. Basu, R. L. Chisholm, R. J. Dodson, E. Hartline, P. Fey, P. D. Thomas, L. P. Albou, D. Ebert, M. J. Kesling, H. Mi, A. Muruganujan, X. Huang, S. Poudel, T. Mushayahama, J. C. Hu, S. A. LaBonte, D. A. Siegele, G. Antonazzo, H. Attrill, N. H. Brown, S. Fexova, P. Garapati, T. E. M. Jones, S. J. Marygold, G. H. Millburn, A. J. Rey, V. Trovisco, G. Dos Santos, D. B. Emmert, K. Falls, P. Zhou, J. L. Goodman, V. B. Strelets, J. Thurmond, M. Courtot, D. S. Osumi, H. Parkinson, P. Roncaglia, M. L. Acencio, M. Kuiper, A. Lreid, C. Logie, R. C. Lovering, R. P. Huntley, P. Denny, N. H. Campbell, B. Kramarz, V. Acquaah, S. H. Ahmad, H. Chen, J. H. Rawson, M. C. Chibucos, M. Giglio, S. Nadendla, R. Tauber, M. J. Duesbury, N. T. Del, B. H. M. Meldal, L. Perfetto, P. Porras, S. Orchard, A. Shrivastava, Z. Xie, H. Y. Chang, R. D. Finn, A. L. Mitchell, N. D. Rawlings, L. Richardson, A. Sangrador-Vegas, J. A. Blake, K. R. Christie, M. E. Dolan, H.
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