PKA, cAMP, and Epac are not required for egress.

(A) Western blots data monitoring egress kinetics of DMSO- and RAP-treated PKAc-HA:loxP and ACβ-HA:loxP schizonts. The slower onset of detection of SERA5 p50 in RAP-treated ACβ-HA:loxP parasites indicates delayed or impaired egress in the absence of cAMP. Blots are representative of two biological repeats, which are both quantified in S4A Fig. (B) Quantification of the proportion of schizonts rupturing in 30-min videos of DMSO- and RAP-treated parasites. For each video, one parasite population (DMSO or RAP) was stained with Hoechst (indicated in blue on the plots). The p-values derive from paired t tests. (C) Quantification of the mean time taken for the DMSO- and RAP-treated parasites from (B) to progress to egress, as measured by visual analysis of the same video microscopy data shown in panel (B). The p-values derive from paired t tests. For all data in (B) and (C), each point is the mean for one population (DMSO or RAP) from a single video (50–100+ schizonts). Ten videos were quantified from at least three independent experiments. (D) Schematic representation of the selection-linked targeted homologous recombination-based approach used to disrupt the PfEpac gene. Lollipops represent translational stop codons. Validation of this line by PCR is shown in S1E Fig. (E) Western blot data indicating normal rupture of PfEpac-deficient schizonts. Data associated with this figure can be found in the supplemental data file (S1 Data). AmpR, ampicillin resistance cassette used for plasmid selection in bacteria; cAMP, cyclic AMP; Epac, exchange protein directly activated by cAMP; hDHFR, human dihydrofolate resistance selectable marker; KO, knockout; NeoR, neomycin resistance selectable marker; PKA, cAMP-dependent protein kinase; p50, processed 50 kDa form; RAP, rapamycin; SERA5, serine repeat antigen 5; T2A, thosea asigna virus 2A peptide.