For IFAs, intracellular tachyzoites grown on a monolayer of HFFs and incubated in the presence or absence of ATc for various periods of time were fixed for 20 min with 4% (wt/vol) paraformaldehyde in PBS, permeabilized for 10 min with 0.3% Triton X-100 in PBS, and blocked with 0.1% (wt/vol) BSA in PBS. The primary antibodies used for detection were anti-TgATRX1 (1/1,000), anti-mitochondrial F1β ATPase (1/1,000) (P. Bradley, unpublished data), anti-TgCPN60 (28 (link)) (1/2,000), anti-TgATG8 (26 (link)) (1/1,000), anti-AMA1 (56 (link)) (1/10,000), anti-TgIMC1 (36 (link)) (1/1,000), anti-GRA3 (55 (link)) (1/500), anti-RON4 (57 (link)) (1/500), and anti-centrin1 (1/500) (I. Cheeseman, unpublished data) antibodies. Staining of both nucleus and apicoplast DNAs was performed with fixed cells incubated for 5 min in a 1-µg/ml DAPI solution. All images were acquired at the Montpellier RIO Imaging facility from a Zeiss Axio Imager Z2 epifluorescence microscope equipped with an ORCA-flash 4.0 camera (Hamamatsu) and driven by the ZEN software (Zeiss). Adjustments for brightness and contrast were applied uniformly to the entire image. For quantification, at least three independent replicates were used.