Western blot analysis in adherent and spheroid cells.

<p>The figure presents a representative experiment obtained with SK-ChA-1. Ad: adherent; Sp: spheroid; TM: trans-membrane form of cadherin E; S: soluble form of cadherin E.</p

Morphology of cultures of the SK-ChA-1 cell line.

<p>Panel A, appearance of the adherent bidimensional culture and, Panel B, appearance of the MCTS (Multicellular Tumor Spheroids). In both instances cells were examined by phase contrast microscopy at 10x magnification.</p

Top canonical pathways identified in cholangiocarcinoma cells EMT.

<p>The graph represents host cell pathways with highest score (y-axis) based on the number of differentially regulated proteins using Ingenuity protein analysis. The bar graphs are the pathways most associated with the proteins altered. The orange line graph shows the ratio of the number of molecules from the differentially expressed proteins in EMT that are in the pathway relative to the total number of molecules in the pathway.</p

2D-PAGE separation of proteins contained in the cell lysates of SK-ChA-1 and MZ-ChA-1.

<p>Proteins were first separated by IEF (pH range 3–10) on a non-linear IEF strip (basic pH at the right) followed by SDS-PAGE in the vertical dimension on a 12.5% gel. Circled protein spots were analyzed by mass spectrometry to compare the relative abundance and results were reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0118906#pone.0118906.t001" target="_blank">Table 1</a>.</p

Differential expression of proteins identified by 2D PAGE.

<p>Following bi-dimensional separation of SKCHA1(SK) and MZCHA1(MZ) cell lysates, the amount of protein present in selected spots of adherent and spheroid cells was determined by densitometry and values expressed as spheroid/adherent ratio (with statistically significant p > 0.05). A ratio >2 indicates that the protein is upregulated during EMT while a ratio <0.5 denotes proteins downregulated during EMT.</p><p>Differential expression of proteins identified by 2D PAGE.</p

Top network functions affected in the EMT process of cholangiocarcinoma cells.

<p>Two relevant networks were generated from EMT-modulated proteins according to the Ingenuity Pathway Knowledge Criteria. A. Cellular compromise, cellular function and maintenance (score = 45). Red, upregulated proteins; green, significantly downregulated proteins; white, proteins known to be in the network but not identified in this study. The color depth indicates the magnitude of the change in protein expression levels. The shape is indicative of the molecular class (i.e protein family). Lines connecting the molecules indicate molecular relationships. Dashed lines indicate indirect interactions, and solid lines indicate direct interactions. The arrow styles indicate specific molecular relationships and the directionality of the interaction. B. Network build up from the three most significant bio-functions (activation Z score >2 or <2): cell survival, synthesis of nitric oxide and migration of cells. The symbols nomenclature is shown in the lower panel.</p

Table_1_A rare loss-of-function genetic mutation suggest a role of dermcidin deficiency in hidradenitis suppurativa pathogenesis.xlsx

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease with a multifactorial aetiology that involves a strict interplay between genetic factors, immune dysregulation and lifestyle. Familial forms represent around 40% of total HS cases and show an autosomal dominant mode of inheritance of the disease. In this study, we conducted a whole-exome sequence analysis on an Italian family of 4 members encompassing a vertical transmission of HS. Focusing on rare damaging variants, we identified a rare insertion of one nucleotide (c.225dupA:p.A76Sfs*21) in the DCD gene encoding for the antimicrobial peptide dermcidin (DCD) that was shared by the proband, his affected father and his 11-years old daughter. Since several transcriptome studies have shown a significantly decreased expression of DCD in HS skin, we hypothesised that the identified frameshift insertion was a loss-of-function mutation that might be associated with HS susceptibility in this family. We thus confirmed by mass spectrometry that DCD levels were diminished in the affected members and showed that the antimicrobial activity of a synthetic DCD peptide resulting from the frameshift mutation was impaired. In order to define the consequences related to a decrease in DCD activity, skin microbiome analyses of different body sites were performed by comparing DCD mutant and wild type samples, and results highlighted significant differences between the groins of mutated and wild type groups. Starting from genetic analysis conducted on an HS family, our findings showed, confirming previous transcriptome results, the potential role of the antimicrobial DCD peptide as an actor playing a crucial part in the etio-pathogenesis of HS and in the maintenance of the skin’s physiological microbiome composition; so, we can hypothesise that DCD could be used as a novel target for personalised therapeutic approach.</p

DataSheet_1_A rare loss-of-function genetic mutation suggest a role of dermcidin deficiency in hidradenitis suppurativa pathogenesis.docx

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease with a multifactorial aetiology that involves a strict interplay between genetic factors, immune dysregulation and lifestyle. Familial forms represent around 40% of total HS cases and show an autosomal dominant mode of inheritance of the disease. In this study, we conducted a whole-exome sequence analysis on an Italian family of 4 members encompassing a vertical transmission of HS. Focusing on rare damaging variants, we identified a rare insertion of one nucleotide (c.225dupA:p.A76Sfs*21) in the DCD gene encoding for the antimicrobial peptide dermcidin (DCD) that was shared by the proband, his affected father and his 11-years old daughter. Since several transcriptome studies have shown a significantly decreased expression of DCD in HS skin, we hypothesised that the identified frameshift insertion was a loss-of-function mutation that might be associated with HS susceptibility in this family. We thus confirmed by mass spectrometry that DCD levels were diminished in the affected members and showed that the antimicrobial activity of a synthetic DCD peptide resulting from the frameshift mutation was impaired. In order to define the consequences related to a decrease in DCD activity, skin microbiome analyses of different body sites were performed by comparing DCD mutant and wild type samples, and results highlighted significant differences between the groins of mutated and wild type groups. Starting from genetic analysis conducted on an HS family, our findings showed, confirming previous transcriptome results, the potential role of the antimicrobial DCD peptide as an actor playing a crucial part in the etio-pathogenesis of HS and in the maintenance of the skin’s physiological microbiome composition; so, we can hypothesise that DCD could be used as a novel target for personalised therapeutic approach.</p

Table_4_A rare loss-of-function genetic mutation suggest a role of dermcidin deficiency in hidradenitis suppurativa pathogenesis.xlsx

Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease with a multifactorial aetiology that involves a strict interplay between genetic factors, immune dysregulation and lifestyle. Familial forms represent around 40% of total HS cases and show an autosomal dominant mode of inheritance of the disease. In this study, we conducted a whole-exome sequence analysis on an Italian family of 4 members encompassing a vertical transmission of HS. Focusing on rare damaging variants, we identified a rare insertion of one nucleotide (c.225dupA:p.A76Sfs*21) in the DCD gene encoding for the antimicrobial peptide dermcidin (DCD) that was shared by the proband, his affected father and his 11-years old daughter. Since several transcriptome studies have shown a significantly decreased expression of DCD in HS skin, we hypothesised that the identified frameshift insertion was a loss-of-function mutation that might be associated with HS susceptibility in this family. We thus confirmed by mass spectrometry that DCD levels were diminished in the affected members and showed that the antimicrobial activity of a synthetic DCD peptide resulting from the frameshift mutation was impaired. In order to define the consequences related to a decrease in DCD activity, skin microbiome analyses of different body sites were performed by comparing DCD mutant and wild type samples, and results highlighted significant differences between the groins of mutated and wild type groups. Starting from genetic analysis conducted on an HS family, our findings showed, confirming previous transcriptome results, the potential role of the antimicrobial DCD peptide as an actor playing a crucial part in the etio-pathogenesis of HS and in the maintenance of the skin’s physiological microbiome composition; so, we can hypothesise that DCD could be used as a novel target for personalised therapeutic approach.</p