Figure 4. Temporal dynamics of pfap2-g transcript levels.

a, Reverse transcription-quantitative PCR (RT-qPCR) time-course analysis of pfap2-g expression in tightly synchronized cultures of the non-transgenic parasites lines F12 and E5. Parasite age is expressed in h post-invasion (hpi). b, Expression of pfap2-g in cultures in which 80 nM ML10 was added at 25-30 hpi, and in control cultures without the inhibitor. At 45-50 hpi ML10-treated cultures contained only mature schizonts, whereas control cultures already contained abundant rings. ML10 was washed out of treated cultures and RNA collected 2 h later, when substantial re-invasion had occurred (“wash +2 h”). Images of Giemsa-stained smears of the different preparations (representative of three independent experiments) are shown. Scale bar, 5 µm. c, Time-course analysis of pfap2-g expression in cultures of the E5-HA-DD line maintained in the absence of Shld (-Shld) or with Shld added at 0-5 hpi. Re-synchronization to a 5 h age window was performed between cycles 1 and 2. In cycle 1, pfap2-g expression was significantly higher in the presence of Shld compared to the -Shld condition at all time points analyzed (p=0.002, 0.002 and 0.027 at 10-15, 20-25 and 30-35 hpi, respectively, using a two-sided t-test with equal variance). In all panels, transcript levels were normalized against ubiquitin-conjugating enzyme (uce). Values are the average of two (c) or three (a-b) independent biological replicates (red dots). Error bars in panels a-b are S.E.M.