PLB activity was expressed as mM of substrate hydrolyzed per minute, per milligram of protein. Total protein concentrations were measured using the Protein Assay kit (Quant-iT – Invitrogen Corp., Carlsbad, CA, USA). Significance tests were carried out comparing each treatment with the control value (100%) using a one-sample Student’s t-test. P < 0.05 was taken as the limit to

indicate significance. Real-time RT-PCR validation of differentially expressed genes The real-time RT-PCR system using SYBR Green detection (Applied Biosystems) was used to analyze gene expression in RNA samples. After treatment with DNase I (Invitrogen Corp., Carlsbad, CA, USA) in the presence of RNase inhibitor see more (Invitrogen Corp., Carlsbad, CA, USA), equal amounts of RNA (1 μg) were reverse transcribed using oligo(dT)12-18

S3I-201 cost primer and submitted to real time PCR. Amplification assays were carried out with a 7900HT Sequence Detection System ABI PRISM instrument (Applied Biosystems, Carlsbad, CA, USA) in 12 μL reactions containing 0.4 μM of each primer (listed in Tables 2 and 3), 6 μL of SYBR Green PCR Master mix (2 ×), and 0.2 μL of template cDNA. After initial denaturation at 95°C for 10 min, amplifications were performed for 40 cycles of: 95°C for 15 s followed by 60°C for 1 min. Table 2 Primers Paracoccidioides brasiliensis used for real time RT-PCR Cluster IDa Geneb Forward primer (5′-3′) Reverse primer (5′-3′) 50 sod3 CTGTTCGCTGGGCTTTGC TCAGTAGTGACGGCTTCCATCAT 1688 icl1 GCTCACCCAGATGGTCAAAT AGTATCCGCATCCGCAATAA 3306 plb1 GCAATGCAAGGGAAGAAAGA CGATCCGAGGAACTCTAACG a ID: identification. b Abbreviations: sod3 (Cu, Zn superoxide dismutase); icl1 (isocitrate lyase); plb1 (phospholipase B). Table 3 Primers for real time RT-PCR to measure gene expression using RNA from alveolar macrophage (MH-S) cells Cluster IDa Geneb Forward primer

(5′-3′) Reverse primer (5′-3′) 272294 Rps9 CGCCAGAAGCTGGGTTTGT CGAGACGCGACTTCTCGAA 21961 nkrf ACCTTTCAACCTACGATGGTCAGA GAGCTCTCACATGGAATTTGGAA 575033 nfkb AGCCAGCTTCCGTGTTTGTT AGGGTTTCGGTTCACTAGTTTCC 104798 tnf -α GTACCTTGTCTACTCCCAGGTTCTCT GTGGGTGAGGAGCACGTAGTC 574821 clec2 CTCTTCTTGGTGGCGTGTGA AACAACCAGCCCCATGGA 3989461 il-1β GTGTGTGACGTTCCCATTAGACA CAGCACGAGGCTTTTTTGTTG 1346060 trl2 AAGAGGAAGCCCAAGAAAGC CGATGGAATCGATGATGTTG 5120996 cd14 CGCAGCCTGGAATACCTTCTA CCGCTTTAAGGACAGAGACTTGATA a ID: identification. b Abbreviations: Protein Tyrosine Kinase inhibitor Rps9 (constitutive ribosomal macrophage gene); nkrf (GSK2245840 supplier NFKappaB repressing factor); nfkb (P50 subunit of NFKappaB); tnf-α (tumor necrosis factor-alpha); clec2 (C- type lectin like receptor); il-1β (Interleukin-1β); trl2 (toll-like receptor 2); cd14 (glycosyl-phosphatidylinositol-anchored glycoprotein). The comparative crossing threshold (CT) method, employing the constitutive ribosomal Rps9 macrophage gene or P. brasiliensis α-tubulin gene, was used in order to normalize the expression value of each gene of interest in the macrophage infected sample compared with the non-infected control.