Flow cytometry analysis

For parasite multiplication assays, synchronous NF54/AP2-G-mScarlet/CK2α-GFPDD parasites were split at 0–6 hpi (0.2% parasitaemia) and cultured either in presence of 675 nM Shield-1 (+Shield-1) or in absence of Shield-1 (−Shield-1) during the whole duration of the assay. Synchronous NF54::DiCre/CK2α-GFP cKO parasites were split at 0–6 hpi (0.2% parasitaemia) and exposed for 4 h to 100 nM RAPA to trigger excision of the pfck2α-gfp gene (DMSO was added to the control population instead of RAPA)^52,54. Subsequently, the RBCs were washed once in PCM/2 mM choline chloride and resuspended in this medium for onward in vitro culture. After 18 h (18–24 hpi) parasite DNA was stained at 37 °C for 30 min using SYBR Green DNA stain (1:10,000, Invitrogen) to determine the starting parasitaemia (day 1). Flow cytometry measurements were repeated on day 3 (generation 2) and day 5 (generation 3) for NF54/AP2-G-mScarlet/CK2α-GFPDD parasites (±Shield-1) and on day 3 (generation 2), day 5 (generation 3), and day 7 (generation 4) for NF54::DiCre/CK2α-GFP cKO parasites (RAPA/DMSO).

To analyse parasite progression through schizogony and merozoite egress, synchronized NF54::DiCre/CK2α-GFP cKO parasites (0–4 hpi) were treated with RAPA or DMSO as explained above. Samples were collected for DNA content analysis starting at 20–24 hpi up to 20–24 hpi in the following generation. For merozoite egress experiments, 1 µM compound 2 (C2) was added to the cultures from 36 to 40 hpi onwards to prevent schizont rupture⁵⁵. At 50–54 hpi to 54–58 hpi, C2-arrested segmented schizont cultures were split and one half was directly inactivated by fixation in 4% formaldehyde/0.0075% glutaraldehyde (C2-arrested control). The other half of the sample was washed once in culture medium to remove C2, resuspended in culture medium, and rotated at 37 °C to allow merozoite egress and invasion for 45 min (replicate 1) and 45 min and 90 min (replicate 2) before samples were fixed in 4% formaldehyde/0.0075% glutaraldehyde. Fixed samples were washed twice in PBS and permeabilized for 15 min in PBS containing 0.1%Triton X-100 and 0.1 mg/ml RNase A. Fixed and permeabilized cells were washed twice in PBS and stained with SYBR Green DNA stain (1:5,000, Invitrogen) for 30 min.

Fluorescence intensities were measured using the MACS Quant Analyzer 10 (200,000 RBCs measured per sample). Data were analysed using the FlowJo_v10.6.1 software. Gating was performed to remove small debris (smaller than cell size), doublets (single measurement event consisting of two cells), and to separate uninfected from infected RBCs (using an uninfected RBC control sample) based on their SYBR green intensity (Supplementary Fig. 4). For the results presented in Supplementary Fig. 7b an additional gate was set to separate mature schizonts from remaining iRBCs based on SYBR green intensity (Supplementary Fig. 8).