Cultured slender or stumpy trypanosomes were differentiated in vitro by the addition of cis-aconitate and a temperature reduction to 27°C. At the times indicated, trypanosomes were analysed for the expression of the fluorescent stumpy reporter GFP:PAD1UTR, as in Figure 5. Slender cells (n=1653) are shown in blue and stumpy cells (n=1798) in red. Data were compiled from five independent experiments, with each time point being analysed in at least two separate experiments. (A) Percentages of PAD1-positive slender and stumpy cells over time. Points indicate the individual experiments for either slender (blue) or stumpy (red) trypanosomes. Point sizes correspond to the total number of cells counted per experiment. These data were fed into a point estimate model, and are shown as solid lines, indicating the predicted percentage of PAD1-positive cells, based on time vs. cell type. Transparent colours indicate the associated 95% confidence bands. The difference between slender and stumpy cells over time was strongly significant (p<0.001). (B, C) Slender and stumpy trypanosomes scored as PAD1-positive or -negative were also stained with DAPI, and the cell cycle position determined based on the configuration of kinetoplast (K) to nucleus (N) at the timepoints indicated. The dividing slender population (B) and dividing stumpy population (C) are shown. As seen, the percentage of PAD1-positive slender cells steadily increased (B, blue) and the percentage of PAD1-negative cells steadily decreased (B, grey). This shows that slender cells can turn on the PAD1 pathway without apparent cell cycle arrest. Although a small portion of the stumpy population was observed to to divide throughout the time points (C, red), cells did not return to a normal cell cycle profile until 48 hr after the addition of cis-aconitate. As the cells became more procyclic, they began to lose their PAD1 signal and an increase in PAD1-negative dividing cells was seen (B, C, grey). Data are shown as mean +/- SD. Points without SD were the result of two measurements at those timepoints.