Figure 5.

(a) Flow cytometry profile of rescue line in comparison with control and HAT4 knockout lines. Promastigotes of Ld1S-neo/hyg/bleo, LdHAT4-KO/bleo and LdHAT4-KO/HAT4-FLAG were arrested at G1/S using 5 mM hydroxyurea and then released into S phase. Time indicated above each histogram indicates the time after release from block. The experiment was carried out three times as detailed in Methods and one dataset is presented here. For each cell type 30,000 events were recorded at every time-point, and data were analyzed using CellQuest Pro Software (BD Biosciences). M1, M2 and M3 gates indicate G1, S and G2/M phases respectively. Percent of cells in G1, S and G2/M at the different time-points are indicated in the upper right-hand corner of each histogram box. (b) Analysis of survival of HAT4-null parasites within macrophages. The number of parasites within macrophages were scored microscopically using DAPI staining and Z-stack image analysis with the help of a confocal microscope. Three replicates of the experiment were separately initiated and carried out in parallel. Mean values are presented in the bar chart. Error bars indicate standard deviation. Student’s t-test was applied to analyze the data. P values obtained: **p < 0.005.