Fig. 9.

Neither apoptosis nor necroptosis is triggered by Candidalysin. a Phosphatidylserine exposure and cell viability of hMDMs infected with C. albicans Wt, re-integrant (ece1Δ/Δ + ECE1) or mutant strain (ece1Δ/Δ) (MOI 10) or treated with synthetic Candidalysin for 5 h were quantified by staining with FITC-Annexin V and PI, respectively. The number of single-stained or double-stained macrophages was evaluated by manual counting of at least 200 macrophages. b Caspase 3/7 activity was assessed by measuring luminescence of hMDMs 7 h post infection with C. albicans Wt or ece1Δ/Δ mutant strain (MOI 10) or co-incubation with Candidalysin. Staurosporine served as a positive control. Shown are relative luminescence values (RLU) after background subtraction. c, d LPS-primed hMDMs were treated with synthetic Candidalysin or Nigericin for 5 h. Selected samples were pre-treated with c the necroptosis inhibitor Necrostatin-1 (Nec-1) or d the actin cytoskeleton inhibitor Cytochalasin D or inhibitor solute control DMSO 1 h prior to administration of synthetic Candidalysin or Nigericin. Macrophage lysis was quantified by measuring LDH release. e LPS-primed hMDMs, mBMDMs or mBMDCs were treated with synthetic Candidalysin or Nigericin for 4–5 h. Selected samples were pre-treated with the potassium channel inhibitor glibenclamide or inhibitor solute control DMSO 1 h prior to administration of synthetic Candidalysin or Nigericin. KCl was added after LPS priming. Macrophage lysis was quantified by measuring LDH release. a Data are shown as mean + SD of two different donors. b–e Values are represented as scatterplot with median of three independent donors or experiments (n ≥ 3). For statistical analysis, a one-way ANOVA with Dunnett’s multiple comparison test was used. ***p ≤ 0.001, significance compared to Candidalysin treatment