Figure 2. Autophagy is induced by Candida but does not play a role in Candida uptake by macrophages and neutrophils.

(a) RAW-Blue cells (macrophage cell line) were co-cultured with Candida labeled with Alexa Fluor-647 (AF-647)(pink) for 0 or 60 min at the 1:1 ratio of Raw-Blue and spore cell. LC3 and nuclei were shown with green and blue, respectively. Corresponding Nomarski images are shown below. Scale bars = 5 μm. (b) Western blotting detection of LC3II in RAW-Blue cells stimulated with C. albicans (1:1 cell ratio) at the indicated time points. (c–e) BMDMs (c) and neutrophils (d) obtained from Atg7 CKO and WT mice were fluorescently labeled and co-cultured with AF-647-labeled Candida (1:5 ratio of host cells and Candida spores) for indicated duration. AF-647-positive cells were determined by flow cytometry. Intensity of AF-647 (Candida) in BMDMs at 30 min was assessed by flow cytometry analysis (e). The histogram is pre-gated on F4/80⁺. (f) To determine macrophage ROS production, dihydroethidium (DHE) staining in BMDMs was evaluated by flow cytometry 2 hrs after co-culture with Candida (1:1 ratio of BMDMs and spores). (g–i) Inhibition of Candida expansion by BMDM (g, h) and neutrophils (i) was evaluated by the method described in the METHODS section. Candida expansion with or without BMDMs was shown at indicated time points (g), and inhibition of Candida expansion was evaluated at 12 hrs after co-culture with host cell culture. (j) Intensity of LC3 signals (green) in a Candida (pink)-engulfed RAW-Blue cell at 60 min after starting co-culture was analyzed along with the yellow line in the panel at right bottom. Scale bar = 2.5 μm. Shown in (a) and (j) are typical examples from 100 cells observed in four independent experiments. Results in (b-i) are representatives of at least two experiments with more than three mice/group. Error bars represent mean ± SD. Student’s t-test was used for statistical analysis. N.S.; not significant. **; p <0.01. ***; p < 0.001.