Controls for lack of D4H toxicity, ketoconazole and terbinafine treatment, and microtubule depolymerization. (A) Expression of mCherry-D4H and deletion of ltc1 do not influence growth capability of S. pombe cells. Serial dilutions of WT and ltc1Δ mutant expressing or not mCherry-D4H were compared for ability to grow at 25°C and 30°C. (B) Expression of mCherry-D4H does not influence cell length and width. Results represent three independent experiments. (C) Ergosterol biosynthesis pathway. (D) Cells treated with 200 mM ketoconazole or solvent (DMSO) for 8 h. (E) Terbinafine inhibits S. pombe growth. WT and ltc1Δ mutant cells expressing or not mCherry-D4H were diluted to OD 0.02 and incubated with 0.1 µg/ml terbinafine for 25 h in EMM-ALU. Afterward, OD was measured. Results represent three independent experiments. (F) Terbinafine does not display cytotoxic effect in S. pombe. Yeast cells were treated with 0.1 µg/ml terbinafine for 16 h at 25°C. Cell viability was evaluated by flow cytometry upon propidium iodide (PI) staining. (G) Microtubule depolymerization has no effect on mCherry-D4H distribution and relocalization. Cells expressing mCherry-D4H and tagged α-tubulin (GFP-Atb2) were incubated in the presence of DMSO (1%), CK-666 (500 µM), or metenzimidazoleazol-2-yl-carbamate (MBC 25 μg/ml), or were pretreated with MBC for 15 min and subsequently treated with CK-666 for 1 h. Arp2/3 inhibition resulted in relocalization of mCherry-D4H signal from the PM toward the cell interior independently of microtubule integrity. Microtubule depolymerization did not trigger relocalization of mCherry-D4H. Error bars show SD. Scale bars, 2 µm.