C. albicans Modulation of Receptor Tyrosine Kinase Signaling

The capacity of the various C. albicans strains to induce phosphorylation of EGFR and c-Met in the presence and absence of inhibitors was determined as described previously (Swidergall et al., 2018 (link)). Briefly OKF6/TERT cells were seeded onto 24 well plates and incubated overnight in KSF medium without supplements. The next morning, the medium was aspirated and replaced with either fresh medium alone or containing gefitinib and/or SGX-523. When inhibitors were used, control cells were incubated with KSF medium containing a similar volume of the DMSO diluent. After 1 h, the cells were infected with 1×10⁶C. albicans germ tubes in the presence of the inhibitors and incubated for 20 min. Next, the medium was aspirated and the epithelial cells were lysed with 2X SDS loading buffer (#BP-111R, Boston Bioproducts, Inc.) containing phosphatase/protease inhibitors (# A32959, Thermo Fisher Scientific), and PMSF (#P7626, Sigma-Aldrich). After denaturing the samples at 90°C for 2 minutes, the lysates were clarified by centrifugation. The proteins were separated by SDS-PAGE and transferred to PVDF membranes. The phosphorylated proteins were detected by probing the membranes with an anti-phospho-c-Met antibody (Tyr1234/1235, #3077, Cell Signaling Technology) or an anti-phospho-EGFR antibody (Tyr1068, #2234, Cell Signaling Technology). Next the blots were stripped and total c-Met was detected with an anti-met antibody (# 8198, Cell Signaling Technology) and total EGFR was detected with an anti-EGFR antibody (#4267, Cell Signaling Technology). The blots were developed using enhanced chemiluminescence, imaged with a digital imager, and quantified using Image Studio Lite software. Each experiment was repeated at least four times.