Fig. 2: Depletion of TbRPA1 induces cellular lethality, abnormalities in cell-cycle progression, and DNA replication.

a TbRPA1 is essential for T. brucei cell viability. TbRPA1 RNAi depletion was induced with the addition of tetracycline and cell growth was monitored by counting cells every 12 for 48 h. WT was used as a control. Three technical replicates were used for each measurement (n = 3). Error bars indicate mean ± SD. An unpaired two-sided Student’s t-test was performed, and statistically significant results were indicated with ****p < 0.0001. b Viability of TbRPA1-depleted trypanosome cells. Cells from each time point in Fig. 2a were examined for viability using the AlamarBlue assay. Percent viability compared to non-depleted cells was determined and plotted. Three technical replicates were used for each measurement (n = 3). Error bars indicate mean ± SD. An unpaired two-sided Student’s t-test was performed, and statistically significant results were indicated with ****p < 0.0001. c Immunoblot controls presenting the changes in protein levels for TbRPA1, γH2A, VSG3, and Tubulin at each time point after depletion. Tubulin serves as a loading control. Three independent experiments were performed with similar results. d Cell-cycle profiles of TbRPA1-depleted cells. Fixed cells were stained with PI and analyzed by flow cytometry. e DNA synthesis assay by BrdU pulse labeling. WT and TbRPA1 KD cells were pulse-labeled with 500 µM BrdU and fixed. Fixed cells were then stained with PI (bulk DNA) and anti-BrdU-Alexa 488 antibody (newly synthesized DNA) and analyzed by flow cytometry. Source data are provided as a Source Data file.