Figure legends
Fig. 1. Localization of P. berghei Scd in the parasite
endoplasmic reticulum. (A ) IFA with asexual blood stage
parasites confirmed Scd expression, which colocalized with the
endoplasmic reticulum (ER) marker Bip. (B ) The expression of
Scd was monitored during liver stage development at different time
points, and we found late expression of Scd at 48 and 55 hpi, which
colocalized with the ER marker BiP. The nuclei were stained with
Hoechst.
Fig. 2. Scd KO parasites display defects in late
liver stage development. (A ) To determine the liver parasite
burden, sporozoites were injected intravenously into C57BL/6 mice,
livers were homogenized at 36, 55 and 72 hpi, RNA was isolated, and
transcripts were quantified using real-time PCR. The 18S rRNA copy
number was determined and normalized to mouse GAPDH transcripts. TheP. berghei 18S rRNA copy number was comparable in WT GFP andScd KO parasites at 36 hpi but significantly lower in ScdKO parasites than in WT GFP parasites at 55 hpi, and at 72 hpi, it was
opposite and found to be significantly higher in KO parasites,
indicating that only WT GFP parasites were able to egress from the
liver. Similar results were obtained in three independent experiments.
Data represent the mean ± SD, n = 5 mice per group. (B ) A lower
level of MSP1 transcripts at 55 hpi in KO-infected liver suggests a
failure in the maturation of parasites. Data are presented as the mean ±
SEM from two independent experiments (significant difference (p=0.0132),
Student’s t test).
Fig. 3. Scd KO liver stages grow normally. (A)
Sporozoite-infected HepG2 cells were fixed at 12, 24, 36, 48, and 62 hpi
and immunostained with anti-UIS4 antibody. Nuclei were stained with
Hoechst. Visually, liver stage growth appeared to be comparable inScd KO and WT GFP parasites. (B, D and F ) The EEF
numbers were found to be normal at 36, 48, and 62 hpi (p=0.3950 at 36
hpi, p= 0.9419 at 48 hpi, and p= 0.6070 at 62 hpi). (C, E and
G ) Determination of EEF size at 36, 48, and 62 hpi. The size of EEFs
was comparable between WT GFP and Scd KO parasites (p=0.5096 at
36 hpi, p=0.6269 at 48 hpi, and p= 0.1642 at 62 hpi). The data were
pooled from three independent experiments. Data represent the mean ± SD.
P values were determined by an unpaired, two-tailed Student’s t test.
Fig. 4. Scd KO parasites arrest late during
liver stage development and do not form hepatic merozoites.(A ) Infected HepG2 cells were harvested at 62 hpi, fixed, and
immunostained with anti-merozoite surface protein 1 (MSP1) antibody, and
DNA was stained with Hoechst. We found normal segregation of nuclei and
formation of merozoites in WT GFP, but it was impaired in Scd KO
parasites. (B ) Counting of nuclei in the EEF revealed impaired
nuclear division in KO parasites (p<0.001, Student’s t test).
The data were pooled from three independent experiments. Data represent
the mean ± SD. (C ) The merosome numbers were counted using a
hemocytometer. Scd KO parasites formed EEFs that were comparable
to WT GFP parasites but failed to form merosomes, a significant
difference (p<0.0001, Student’s t test).
Fig. 5. Lack of Scd leads to defects in organelle morphology and
biogenesis. (A ) Infected HepG2 cells were fixed at 62 hpi and
immunostained with apicoplast marker anti-ACP antibody. WT GFP showed
normal branching of the apicoplast, which was impaired in Scd KO
parasites. (B ) Immunostaining of EEFs with an ER marker
anti-Bip antibody revealed impaired branching in Scd KO
parasites.