Journal: Computational and Structural Biotechnology Journal

Article Title: Comparative host transcriptome in response to pathogenic fungi identifies common and species-specific transcriptional antifungal host response pathways

doi: 10.1016/j.csbj.2020.12.036

Figure Lengend Snippet: Comparative metabolic shift of host cells upon fungal stimulation: R. oryzae induces a dysfunctional glycolysis (A-B) After 12 h of incubation with A. fumigatus , C. albicans and R. oryzae , PBMCs from healthy volunteers were harvested and Oxygen Consumption Rate (OCR) (A) and Extracellular Acidification Rate (ECAR) (B) were measured by Seahorse XF e96; Basal respiration, ATP production, Maximal Respiration, Spare Respiration Capacity (SRC), Proton (H + ) leak (A) and Basal ECAR, Glycolysis, Glycolysis, Glycolytic reserve (B) are represented (mean ± SEM, n = 3 donors, with 4–5 technical quadruplicates per donor, Student’s t test between R. oryzae - stimulated and unstimulated cells). (C) Heatmap of transcriptional expression of glycolytic genes extracted from the RNA-Seq dataset of PBMCs stimulated with A. fumigatus , C. albicans and R. oryzae for 4 h and 24 h compared to unstimulated cells (RPMI); the color indicates the Log 2 FC (Shades of red indicate upregulation, while shades of green downregulation; the white * indicates a corrected p- value of < 0.05, see legend. (D) Expression of HK1 and HK2 measured by qRT-PCR in PBMCs from healthy volunteers (n = 6 donors) under Tunicamycin-induced ER stress for 4 h, as compared with untreated cells. Log-fold changes are expressed as the ratio of gene expression, after normalization to β-actin. (E) ROS production measured in PBMCs from healthy volunteers pre-incubated with A. fumigatus , C. albicans and R. oryzae for 12 h in the presence of 10% serum and then re-stimulated with serum opsonized Zymosan. Measurements (ROS induction, RLU/sec) were taken within 1 h in intervals of 2.23 min and are reported as Area under curve (AUC) (mean ± SEM, n = 3 donors, with 4–5 technical quadruplicates per donor, One way ANOVA test with Dunnett’s multiple correction test). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using Cell Mito Stress test (for OCR) kit and the Glycolysis Stress test (for ECAR) in an XFe96 Analyzer (Seahorse Bioscience), with final concentrations of 1 µM oligomycin, 1 µM FCCP, and 1.25/2.5 µM rotenone/antimycin A for the Mito Stress test, and 11 mM glucose, 1 µM oligomycin, and 22 mM 2-DG for the Glycolysis Stress test.

Techniques: Incubation, Expressing, RNA Sequencing Assay, Quantitative RT-PCR