Figure 2 Data

Figure 2. Using in vitro timelapse microscopy to visualize the melanin-based immune response. (A) Using a developed timelapse microscopy protocol, we were able to record the dynamics of hemolymph melanization in response to C. neoformans from 0 (B) up to 24 hours (C). We can then use particle measurement software to visualize and quantify the melanization reactions over time (D), representative of three biological replicates. This method can be used to gain insight into how different virulence factors, such as laccase, influence the melanization response, where we see that the laccase knockout mutant causes less of a melanization response without much change in time until onset of melanization (E), represented by three biological replicates, and normalized to the wildtype control melanization area at 360 minutes. (F) Overall, the lac1∆ triggers ~40% as much of a melanization immune response compared to the WT (one sample t-test, p = 0.0028, t = 18.78, df =2). (G) Isolated fungal melanins are associated with activation of the insect melanin based immune response, whereas the heat killed acapsular cryptococcal cells are not, as represented by 3-6 biological replicates. (H) Additionally, we can compare the activation of the melanin-based immune response from different fungal species such as C. neoformans and C. albicans, which strongly activates the melanization immune response, represented by 3-6 biological replicates. (B-D, G) are representative images and graphs from three biological replicates. Error bars indicate Standard Deviation. (D, E, G, H) Solid lines indicate mean melanization, and light colored points indicate individual replicate data points. Scale bars represent 50 µm.图2 利用体外延时显微镜（in vitro timelapse microscopy）可视化基于黑色素的免疫应答。(A) 借助优化后的延时显微镜实验方案，我们可记录新型隐球菌（C. neoformans）刺激下血淋巴黑色素化的动态过程，观测时长从0时刻（B）延伸至24小时（C）。随后可通过粒子测量软件可视化并定量不同时间点的黑色素化反应（D），该结果代表3次生物学重复。本方法可用于解析漆酶（laccase）等不同毒力因子对黑色素化应答的调控机制：我们观察到，漆酶敲除突变体引发的黑色素化应答较弱，且黑色素化启动前的时间无显著变化（E），该结果对应3次生物学重复，数据以360分钟时野生型（WT）对照的黑色素化面积进行标准化。(F) 总体而言，与野生型（WT）相比，lac1Δ突变体触发的黑色素化免疫应答仅为其约40%（单样本t检验（one sample t-test）：p=0.0028，t=18.78，df=2）。(G) 分离得到的真菌黑色素可激活昆虫基于黑色素的免疫应答，而热灭活的无荚膜隐球菌细胞则无此效应，该结果对应3~6次生物学重复。(H) 此外，还可比较不同真菌物种（如新型隐球菌与白色念珠菌（C. albicans））引发的黑色素化免疫应答激活情况，其中白色念珠菌可强烈激活该免疫应答，该结果对应3~6次生物学重复。(B~D、G) 为3次生物学重复的代表性图像与统计图，误差棒代表标准差（Standard Deviation）。(D、E、G、H) 中实线代表黑色素化水平均值，浅色散点代表单次重复的实验数据点，比例尺（scale bar）为50 µm。