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. 2015 Nov 30;14(12):1228–1239. doi: 10.1128/EC.00147-15

FIG 5.

FIG 5

Assessment of hyphal branching patterns of tetR-SEC15. (A) Gene expression was repressed by incubating strains for 2 h in YPD with or without DOX; next, filamentation was induced in YPD–20% FCS, and the number of yeast and filamentous cells was determined after 3 h. There was a significant increase in yeast cells in tetR-SEC15 (grown with DOX) and tetR-SEC6 (grown with DOX) compared to controls (P < 0.001). Experiments were performed in triplicate independently, and 100 cells were counted in each experiment. (B) Gene expression was first repressed by incubating strains for 2 h in YPD with or without DOX; then, filamentation was induced in YPD–20% FCS, and the number of lateral branches was determined. Experiments were performed in triplicate, with 100 cells counted per treatment per experiment. Only cells that formed filaments were included for this analysis. Strain tetR-SEC15 grown with DOX (+DOX) had an increased number of branches compared to control strains (THE1-CIp10 with or without DOX and tetR-SEC15 without DOX), and strain tetR-SEC6 had a decreased number of branches compared to control strains (THE1-CIp10 with or without DOX and tetR-SEC6 without DOX). The asterisks indicate statistical significance compared to the controls (P < 0.001). (C) After induction of filamentation, cells were stained with calcofluor white and visualized using fluorescence microscopy (magnification, ×63) using a DAPI (4′,6-diamidino-2-phenylindole) filter. Arrows indicate the number of branches present in each strain. The tetR-SEC15 strain grown with DOX had an increased number of branches; in contrast, we have previously shown that the tetR-SEC6 strain grown with DOX had a decreased number of branches (6). Scale bar, 10 μm. (D) After induction of filamentation, the number of lateral branches was determined in each strain and categorized as 0 to 1 (0-1), 2, 3, or >3 branches. Experiments were performed in triplicate, with 100 cells counted per treatment per experiment. Strain tetR-SEC15 grown with DOX had an increased number of branches compared to its controls (THE1-CIp10 with or without DOX and tetR-SEC15 without DOX). The asterisk indicates statistical significance compared to the controls (P < 0.05).