Fig. 7: In vitro BBB transmigration and adhesion assays for C. neoformans phosphatases.

a In vitro BBB migration and b human brain microvascular endothelial cell line (hCMEC/D3) adhesion assay of pathogenicity-related phosphatases. Right: y-axis indicates trans-endothelial electrical resistance (TEER). Data plots represent individual data from three independent experiments (n = 3). Data presented as mean values ± SEM. Significant differences were calculated by two-tailed (unpaired) Student’s t-test comparing wild-type (WT) and each phosphatase deletion mutant. c Host-mimic condition (HMC)-mediated induction of brain infection-related genes in wild-type (WT) and phosphatase deletion mutant strains. Gene expression was determined by quantitative RT-PCR with cDNA synthesized from total RNA prepared from cells shifted from basal condition (YPD at 30 °C, grey shaded) to HMC (RPMI with 10% foetal bovine serum at 37 °C under 5% CO₂) and further incubated for 3 h (red shaded). Fold-change of gene expression was calculated relative to basal expression levels of each gene in WT. Data from three independent experiments (black dots) are presented as mean values ± SEM. Statistical significance between basal and HMC was determined by two-tailed (unpaired) Student’s t-test (*P < 0.05; **P < 0.001; ***P < 0.0001). d Functional protein association network analysis of BBB crossing-related TFs, kinases, and phosphatases of C. neoformans predicted by STRING (http://string-db.org) with protein sequences obtained from FungiDB (https://fungidb.org/fungidb/). Images were drawn with Cytoscape v3.7.2. Dotted lines: groups of genes involved in purine metabolism (purple), RNA processing (red), and glucose sensing (orange).