Extended Data Figure 3: Microarray analysis of P. berghei parasites under CR.
a, Schematic representation of parasite sample preparation for microarray time-course analysis. P. berghei ANKA parasites collected with 4 h intervals 30 h after intravenous injection of purified schizonts into AL and CR mice. b, Microscopy analysis of parasite size (as proxy for parasite age) in the samples used in the microarrays show no apparent morphological differences in parasite development under AL and CR across the different time-points. The parasite area (a.u., arbitrary units) is defined by the Giemsa staining on thin blood smears33 and was scored using ImageJ. Histograms of parasite size distribution (3 mice per group per time point). The total number of parasites analysed are as follows: 6 h, AL n = 150, CR n = 143; 10 h, AL n = 158, CR n = 159; 14 h, AL n = 158, CR n = 157; 18 h, AL n = 180, CR n = 121). The indicated time-points correspond to the parasite developing time after RBC re-invasion, in the second cycle. c. Scatter plots of log2 fold change (y axis) and mean expression levels (x axis) of parasites in CR versus AL (CR/AL) of 3 mice per group at the indicated time-points. Genes differentially induced or repressed in CR (with log2(fold change) of 1 and FDR adjusted P < 0.01) are highlighted in red and blue, respectively. The number (and relative percentage) of genes altered for each time-point is given in the graphs in red (induced) and blue (repressed). d, Correlation plot between microarray and qPCR analysis for the 14-h samples. 20 genes were selected on the basis of the highest fold-changes, analysed by qPCR and compared to the values obtained in the microarray analysis. Validation rate was 80%. Values are mean of 3 mice per group. The list of genes analysed and their fold-changes in qPCR and microarray are provided in the Source Data. e, qPCR analysis of repressed genes in independent biological samples collected at 14 h (AL n = 4, CR n = 3). Each circle represents one mouse. Gene IDs are shown without the ‘PBANKA_’ prefix. Microarray hybridization was performed one time and confirmed by qPCR for a subset of genes in the same RNA samples (d), as well as independently collected samples (e). f, Gene ontology enrichment analysis (Molecular Function) of the genes showing significant alterations for each time point using PlasmoDB (http://www.plasmodb.org) tools and considering Benjamini–Hochberg adjusted P <0.05. The graph highlights the top four of terms with highest significance for each time point and/or terms that appear more than once. Red, induced; blue, repressed. The full list of terms (including biological process analysis) for each time point are provided in the Source Data.
