Fig. 1
Screening and validation of Sel1 as a SCP of C. albicans. a Schematic presentation of in silico screening of SCPs in C. albicans. b C. albicans SC5314 cells were incubated with Raw 264.7 cells, and the expression levels of SCPs were analyzed by real-time quantitative PCR (qRT-PCR). c Bone marrow-derived macrophages (BMDMs) were infected by C. albicans harboring SEL1-HA. The culture supernatants (Culture Sup.) were collected, centrifuged, and filtered with 0.22 μm column prior to western blotting, while the remaining BMDMs and fungal cells were washed with water and harvested by centrifugation for RNA or protein preparation, respectively. SEL1 (SCP6) RNA levels (top panels) and Sel1-HA (Scp6-HA) protein levels (bottom panels) were assessed by RT-PCR or western blotting. d Sequence alignment of Sel1 homologs in Candida species. N-terminal signal peptide is indicated by red arrow. The cysteine residues are shown in red box, and the conserved cysteines are marked by red asterisks. e–g C. albicans cells grown in various media (6 h) as indicated were harvested and SEL1 expression was analyzed by qRT-PCR at 25 °C for f, g. YPD yeast extract peptone dextrose medium, DMEM Dulbecco’s modified Eagle's medium, 1640 Roswell Park Memorial Institute 1640 medium, SCD synthetic complete dextrose, SCLD SCD medium with low dextrose, SCD-aa SCD medium without amino acids, SCDLA SCD medium with low ammonium. The ingredients of each medium can be found in the Methods section. h C. albicans (SEL1-HA) were grown in various media as indicated for 12 h. The culture supernatants (Culture Sup.) were collected as in c, whereas the fungal cells were collected and lysed for protein lysates. The Sel1-HA proteins in the supernatants and cell lysates were detected by western blotting and quantified over histone H3 (loading control). Data represent the average expression levels of two independent experiments (e–g). Bars, mean ± SEM. Data are representative of at least two (b, c, h) independent experiments each with similar results. Data relative to RNA level in the un-induced C. albicans cells (b, d–f); *p < 0.05, **p < 0.01, ns: no significance, by Student’s t-test
