Fig 1: Characterization of HAS enzyme expression at the tissue level in MPM sections and in MPM cells. (A–C) Immunohistochemical analysis of HAS enzymes in MPM tissues. MPM sections were probed for the detection of HAS1 (A), HAS2 (B) and HAS3 (C) expression at the tissue level, showing cytoplasmic and membrane presence of all the three HAS isoforms within the tumor region. Staining was revealed using a polymer detection kit (Novocastra, Leica Biosystems) and the AEC (3-amino-9-ethylcarbazole, Dako, Denmark) or DAB (3,3′-diaminobenzidine) substrate chromogen. Slides were counterstained with Harris Hematoxylin (Novocastra, Leica Biosystems). Magnification, 200×, scale bar, 100 µm. (D) Basal mRNA expression level of HAS enzymes in MPM primary cells using quantitative real-time PCR. Higher expression of HAS1 and HAS2 was highlighted. TATA box-binding protein (TBP) was used as a housekeeping gene to normalize gene expression results. Data were expressed as the means of experiments performed on eight different MPM populations in triplicates ± SEM. (E) Basal protein expression level of HAS enzymes in MPM cells using cytofluorimetric analysis. MPM cells were stained for HAS1, HAS2 and HAS3 and the fluorescence intensity of the cells incubated with primary antibodies (green line) was compared with unrelated staining (black line). All three enzymes were expressed by MPM primary cells. Basal protein expression level of HAS enzymes was also evaluated in NCI-H28, a human mesothelioma cell line. HAS enzyme expression in NCI-H28 cells appeared comparable to the one of MPM primary cells.