For RT-PCR reactions, cDNA was synthesized using RevertAid™ (Fermentas). In all cDNA synthesis reactions, 1 μg of total RNA (adjusted with Nanodrop 1000) was used. All PCRs were performed as 30 cycles of 95 °C for 1 min, 58 °C for 30 s, and Nutlin-3a cell line 72 °C for 30 s. The A. fumigatus actin fragment (500 bp) was amplified as a loading control during all RT-PCRs. Constructs were prepared to facilitate homologous recombination using Nce102 flanking regions surrounding a pyrG marker (Fig. 1a). A 4-kb fragment containing the entire Nce102 coding
region with upstream and downstream flanking regions was cloned into the pGEM-Teasy vector. From this vector, a 1.8-kb 3′ flanking region of the gene was amplified using primers NCE_KO3 and NCE_KO4 containing EcoRI and SalI sites, respectively (Table S1, Supporting information). This fragment was subsequently cloned into EcoRI/SalI site of pGEM-Teasy vector, yielding pNCE-ko1 plasmid. Likewise,
primers NCE_KO5 and NCE_KO6 containing NotI and EcoRI sites were used to generate an approximately 1.8-kb 5′ flanking region of the gene, which http://www.selleckchem.com/products/ch5424802.html was then cloned into NotI/EcoRI site of pNCE-ko1. To prepare the final construct, pNCE_KO, the A. fumigatus pyrG gene with its own promoter and terminator was cut from a previously prepared pMOD-pyrG plasmid using EcoRI and cloned into EcoRI site of pNCE_ko1 (Fig. 1b). To generate the NCE-EGFP fusion construct, the full-length AfuNce102 cDNA was prepared by RT-PCR using primers NCE_F1 and NCE_R1 containing BglII and HindIII restriction sites, respectively (Table S1). This fragment was subsequently cloned into a BglII/HindIII digest of pGEM-EGP plasmid resulting in the pNCE-EGFP plasmid (Fig. 1b). For the complementation study, a 3.5-kb PCR product containing AfuNce102 and its 5′ and 3′ flanking regions was amplified using primers NCE-F2 and NCE_KO2 (Table S1). The resulting fragment along with plasmid pAN7.1 was used in a co-transformation reaction to transform
the AfuNce102 deletion strain. Mycelia were visualized using a Jenus fluorescence microscope. Digital images were acquired by an INFINITY lite digital camera (Lumenera, Canada) and were prepared using Adobe Photoshop cs version 8.0. Conidia of NCE-EGFP-expressing strain were inoculated Plasmin in maltodextrin medium (1%) on coverslips and incubated at 37 °C for 16 h. The EGFP fluorescence was directly observed using a standard FITC filter. For ER staining, ER-Tracker™ Red dye (Invitrogen) was used at a final concentration of 1 μM in PBS. The strain was grown on a coverslip covered with dye solution for 30 min at 37 °C and washed briefly in PBS before being observed under the microscope equipped with a Rhodamine filter. To stain the nuclei, the mycelia were grown on coverslips as previously described and covered with a 1 μg mL−1 DAPI solution (Sigma) for 30 min at room temperature. After washing in PBS, the stained mycelia were visualized using a standard DAPI filter.