<i>hsp21</i>Δ/Δ exhibits reduced invasive growth and hyphal formation.

<p>(A) Formation of hyphae was induced by embedding fungal cells in YPS (2% saccharose) agar or by plating them on solid water agar supplemented with 10% fetal bovine serum, SLAD agar or on solid Spider medium. Serum agar plates were incubated for 2, SLAD agar plates for 4, and Spider agar plates for 10 days at 37°C. Embedded plates were incubated at 25°C for 5 days. Experiments were performed twice in duplicate. Representative pictures are shown. Scale bar: 100 µm. (B) Hyphal elongation in RPMI1640 and 10% serum. Wild type, <i>hsp21</i>Δ/Δ mutant or <i>hsp21</i>Δ/Δ::<i>HSP21</i> complemented mutant cells were grown overnight in SD medium. After washing twice with water, 10⁴ cells were incubated in RPMI1640 or water supplemented with 10% serum in 24-well cell culture plates at 37°C for 4 hours in the presence of 5% CO₂. Hyphal lengths were then determined using an Inverse microscope (Leica). Results are the mean ± SD of two independent experiments, each performed in duplicate with the length of at least 100 cells measured per strain and experiment. *P<0.0001 compared with the wild type and <i>hsp21</i>Δ/Δ::<i>HSP21</i> complemented strain. Pictures of representative hyphae were taken using a 40x-magnification. Scale bar: 10 µm. (C) Hyphal formation on epithelial monolayers. TR146 epithelial cells were cultured to confluency and infected with <i>C. albicans</i> cells for three hours. Fungal cells were then stained with Calcofluor white (stains invaded and non-invaded fungal elements) and hyphal lengths were determined by fluorescence microscopy. Results are the mean ± SD of two independent experiments, each performed in duplicate with the length of at least 200 cells measured per strain and experiment. *P<0.0001 compared with the wild type strain. (D) Representative pictures of wild type and <i>hsp21</i>Δ/Δ hyphae are shown. Scale bar: 20 µm.