Figure 2.

Detection of melanin-deficient or heat-killed resting A. fumigatus conidia by BMDMs does not induce mitoROS or alter the activity of mitochondrial complexes I and II, and mitochondrial transmembrane potential. (A) Microscopic analysis of mitoROS in BMDMs. BMDMs were left untreated (Control) or infected with conidia lacking DHN melanin (pksp) or heat-killed (HK) resting A. fumigatus conidia for 2 h. Cells were stained with mitoSOX, fixed and subjected to imaging. The scale bars are 5 µm. (B) The abundance of mitoROS in BMDMs was determined by image analysis of MitoSOX-stained BMDMs with ImageJ software. A relative corrected total cell fluorescence (CTCF) was calculated in relation to an average of CTCF measured in untreated (Control) BMDMs. (C) Spectrophotometry analysis of enzyme activities of mitochondrial respiratory chain complex I (CI), complex II (CII), and citrate synthase (CS). BMDMs were left untreated (Control) or exposed to resting or heat-killed resting conidia for 2 h. Enzymatic activity measured in untreated BMDMs was set as 100% in each replicate. The activity of citrate synthase was measured to assess quality of mitochondria. (D) Measurements of mitochondrial membrane potential in BMDMs by detecting fluorescence of MitoView633. BMDMs were exposed to pksp resting conidia. MitoView633 fluorescence was measured after incubation overnight. Data are from two (B, D), or three (C) independent experiments. “n” indicates number of cells used for quantification; bars indicate means and standard errors. Differences between groups were evaluated with the one-way ANOVA.