The quantitative level of PRDM1α mRNA was normalized to β-actin using the cycle threshold (Ct) method (2-△△Ct method). For each sample, 3 independent experiments were made with triplicates for each experiment. Samples from plasma cell myeloma and tonsil were used as positive controls for PRDM1α FK228 purchase mRNA detection. ISH detection ISH for miR-223, miR-886-3p, and miR-34c-5p was performed for 31 EN-NK/T-NTs, 10 peripheral T-cell lymphomas, and 13 inflammatory nasal mucosa specimens. The presence of NK cells within the inflammatory nasal mucosa specimens were identified

by CD56 immunostaining. Probes labelled with a locked nuclear acid (LNA)™ probe for miR-223, miR-886-3p, and miR-34c-5p were designed and generated by Bio Perfectus Technologies (Jiang-su, China) according to sequences in the miRbase (Table 1). DMXAA Table 1 Sequences of in situ hybridisation probes for miR-223, miR-886-3p,

and miR-34c-5p miRNA MiRbase no. Genomic location Probe hsa-miR-223 MIMAT0000280 Xq12 5′-TGGGGTATTTGACAAACTGACA-3′ hsa-miR-886-3p MIMAT0004906 5q31.1 5′-AAGGGTCAGTAAGCACCCGCG-3′ hsa-miR-34c-5p MIMAT0000686 11q23.1 5′-GCAATCAGCTAACTACACTGCCT-3′ The ISH assays for miRNAs were performed as follows: FFPE tissues were routinely deparaffinised in xylene and rehydrated with an ethanol gradient, treated with 1 mg/ml Proteinase K for 10 min at 37°C, fixed with 4% formaldehyde for 10 min, and then dehydrated in ice-cold 90% ethanol. A 20-μL volume of hybridisation mixture consisting of 2 μL of the indicated LNA™ probe and 18 μL of a solution of 200 μg/mL salmon sperm DNA, 1 mg/mL dithiothreitol (DTT), 50% formamide, 2× Denhardt’s, 1 mg/mL

polyglucosan, and 2× saline-sodium citrate (2× SSC) was applied to each slide. The hybridisation reactions were performed overnight at 42°C in a humidified chamber. The sections were stringently rinsed 3 times for 15 min each in 2× SSC at 37°C, and endogenous peroxidases were blocked with 10% H2O2 for 20 min. After 2 washes in 1× PBS for 10 min, the slides were blocked with goat serum (1:100) for 30 min. The slides were then incubated with mouse anti-digoxin antibody for 20 h at 4°C. The slides were washed twice with 1× PBS, incubated with polymer auxiliary agent for 30 min, and washed with 1× PBS (-)-p-Bromotetramisole Oxalate for 10 min. Goat anti-mouse secondary antibody was added to the slides. After 2 washes with 1× PBS, DAB staining was performed. miR-223-, miR-886-3p-, or miR-34c-5p-positive EN-NK/T-NT tissue was used as a positive control for miR-223, miR-886-3p, or miR-34c-5p staining, respectively. For negative control samples, the hybridisation reactions were performed with a sense probe. Cytoplasmic staining was interpreted as miRNA expression, and positive expression was defined as staining of 10% or more of the cells in each tumour. The grading was semi-quantitatively estimated as follows: negative (0% to <10%), weak (10% to ≤50% positive cells), or strong (>50% to 100% positive cells).