blocking reagent; Goat anti-rabbit IgG:HRP, abcam, 1/1000 in blocking reagent. Determination of Relative Protein Amounts after Western Blotting In most cases the differences in protein expression, analyzed by western blotting, were visible to the naked eye. Nevertheless, BIORAD Image Lab 3.0.1 software was used to MedChemExpress 480-44-4 determine the relative protein amounts of the different cell lines by computationally normalizing the target bands to the loading control bands in all samples and then calculating the relative differences between the target bands of the determined cell lines. Enzyme-linked Immunosorbent Assay KEB7 vs. Concentrations were measured in ng/ml or pg/ml. Results for patient cell lines are given as fold expression compared to NEB-1 wild-type cells. Results for patients blister fluids are given as concentrations. doi:10.1371/journal.pone.0070123.t005 7 Therapeutic Targets in Dowling-Meara Cell Lines Statistical Analysis For SQRT-PCR and QuantikineH ELISA experiments, a ‘Students t-test was performed to determine statistical significance with parameters “two-tailed”and “unpaired”. The number of repeats and p-values are given in detail in every figure legend. Results Microarray Analysis Revealed Differentially Expressed Genes in the EBS-DM Cell Lines KEB-7 and EBDM-1 Our group recently demonstrated, that IL-1b is a critical determinant of the phenotype of the EBS-DM patient cell lines KEB-7 and EBDM-1 by way of activating 
the JNK stress pathway. We also showed in the same study that these cells exhibit an invasive phenotype in MatrigelTM invasion chambers. Based on that information, we expected to observe altered gene expression profiles for KEB-7 and EBDM-1 cells compared to NEB-1 wild-type keratinocytes. To identify differentially regulated genes, we performed a whole-genome microarray analysis and compared the transcriptome levels of KEB-7 and EBDM-1 cells to NEB-1 cells. The DAVID bioinformatics tool was used to identify regulated genes that are relevant to the disease pathomechanism, especially regarding blister formation and invasiveness, and the genes were grouped into functional classes. Six major functional classes were defined: 1) kallikrein-related peptidases, 2) matrix metalloproteinases, 3) factors related to actin cytoskeleton dynamics, 4) cytokeratins, 5) 8901831 title=’View abstract’ target=’resource_window’>14557281 junction proteins, and 6) chemokines. Genes were chosen as targets for further analysis even if they were regulated in only one of the two EBS-DM cell lines. We used SQRT-PCR to confirm the microarray data at the gene expression level as well as western blot and ELISA to confirm expression at the protein level. KLK5 and KLK7 showed the highest increase in gene expression in both of the investigated EBS-DM cell lines. Both KLK5 and KLK7 are known to contribute to skin desquamation in the stratum corneum by degrading desmosomal proteins. The increased expression of KLK5 and KLK7 was confirmed by SQRT-PCR for both KEB-7 and EBDM-1 cells. Western blot analysis doi:10.1371/journal.pone.0070123.t006 Therapeutic Targets in Dowling-Meara Cell Lines showed elevated amounts of KLK5 and KLK7 protein in the cell culture supernatant when compared to NEB-1 cells. To evaluate the in vivo situation, we analysed the blister fluids of EBS patients vs. healthy controls. KLK5 and KLK7 protein levels showed no significant differences between patients and controls, as analysed by ELISA and western blot, respectively. Expression of Matrix Metalloproteinase-9 was Increased in EBS-DM Cell Line
Posted inUncategorized