Figure 1 Data

<strong>Figure 1.</strong> <strong>The melanin-based immune response is implicated in killing </strong><em><strong>C. neoformans</strong></em><strong> during infection.</strong> (A) During infection of G. mellonella, melanized nodules are formed within the hemolymph, which encapsulate C. neoformans. Melanin pigment produced by the fungus, is located typically within the cell wall, while the melanin produced by the insect during encapsulation within nodules is found around and within the fungal polysaccharide capsule. (B) GFP fluorescence is lost in heat killed C. neoformans cells expressing GFP under an actin promoter. Data represents counts of 1,640-1,750 single cells from two separate biological replicates. (C) Using a GFP-expressing strain of C. neoformans, we can visualize cell viability within the nodule, with living fungi fluorescing green and dead cells having no signal. (D) Melanin-encapsulated fungi are statistically less likely to have a GFP-positive signal at 24 h after infection at 30˚C, and at 72 h post infection at room temperature and 30˚C (Chi-squared test, p&lt;0.00001, p=0.00004, and p=0.0001, respectively) indicating that melanin-encapsulation is associated with fungal death. Numbers within bars represent the number of fungi enumerated in that condition. (E) In GFP-positive cells, the brighter fluorescence signal correlates with little to no melanization, with no cells that have strong GFP signals and large amounts of melanin encapsulation (n = 40). Each point represents data from one GFP-positive fungus within a nodules from three biological replicates. (F) Propidium Iodide viability staining does not appear to penetrate the inner space of the nodule where the fungus is located, but the staining does show that the hemocytes surrounding the fungus are not viable. (G) Colony forming units (CFUs) correlate directly to melanin inhibition using PTU (inset) in vitro, which indicates that melanin has a role in controlling growth of C. neoformans. Line represents the mean and error bars indicate 95% Confidence Interval of four independent biological replicates with 10-12 total technical replicates. (H) CFUs also inversely correlated to the Mean Gray Value of these wells, meaning that the darker wells with more melanization had higher CFUs recovered compared to the melanin-inhibited wells (Simple Linear Regression, p&lt;0.00001, F= 40.59). Each data point indicates measurements from individual wells from across three biological replicates, dotted lines represent the 95% confidence interval of the linear regression. (A-C,F) Data shown is representative of three biological replicates. All experiments performed in biological triplicate, with (G) representing 5 biological replicates. Scale bars represent 10 µm.