P.Figure 3A, B) Percentage of hepatocytes with schizonts (+/- s.d.) and (B) median size of schizonts (+/- s.d.) for NF54 (blue), NF135 (green), and NF175 (purple) on days 3, 5 and 7 p.i. with sporozoites to hepatocyte ratio of 1:1. Three biological replicates were performed with two technical replicates in each. The mean number of schizonts for the replicates per biological replicates per strain were used for the two-way RM ANOVA test, followed by a Tukey's multiple comparison test. (Mean schizonts per well +/- SDS. NF54 D3 302.5 ±55.477; D5 187.8±61.087; D7 115±29.360. NF135 D3 1555.5±340.4; D5 1284.7±355.4; D7 1138±251.6. NF175 D3 1738±172.679; 1571.8±84.101; 1212.2±206.7). At least 100 schizont sizes were measured for each of three biological replicates and the median is plotted above as the size distribution was not of normal distribution via the D'Agostino and Pearson normality test. The median size per strain for three biological replicates were used in a RM ANOVA test followed by a Tukey's multiple comparison test. Data information: * p <0.05; ** p < 0.005; *** p <0.001; **** p <0.0001 C) Representative immunofluorescence images showing the different sub-population of infected fPHH: hGK (red), Dapi (blue) and phalloidin (cyan). Parasites are stained with PfGAPDH (green). Images taken on the Leica High Content at 20x magnification and then cropped in Adobe Photoshop. D) Percentage schizonts (from total number of schizonts in a well +/- s.d.) located in the different hepatocyte sub-populations by NF54, NF135, NF175 on day 3 p.i.. Each dot represents a biological replicate. For each parasite line, at least 100 schizonts were characterized from 2x 96 wells for the final percentage. The values from three biological replicates were used in a RM ANOVA test followed by a Tukey's multiple comparison test. Percentage of schizonts in Z1/hGK++/hGS- hepatocytes is not different between the three parasite strains. Percentage of schizonts in Z2/hGK+/hGS- for NF54 is significantly different from NF135 (p = 0.0015) and NF175 (p = 0.0015). Percentage of schizonts in Z3/hGK-/hGS+ hepatocytes for NF54 is significantly different from NF135 (p = 0.0002) and NF175 (p = 0.0.0002). Data information: * p <0.05; ** p < 0.005; *** p <0.001; **** p <0.0001 E) Percentage (+/- s.d.) of Z1/hGK++/hGS- (black), Z2/hGK+/hGS- (dark grey) and Z3/hGK-/hGS+ (light grey) hepatocytes that are infected with different Pf strains on day 3 p.i.. This is calculated by dividing the number of schizonts by the actual number of hepatocytes in each zonal subpopulation. Each dot represents a different liver donor (same donors as used in Figure 2D). The values from three biological replicates were used in a RM ANOVA test followed by a Tukey's multiple comparison test. The percentage of Z1/hGK++/hGS- and Z3/hGK-/hGS+ hepatocytes infected with parasites were different for NF135 ( p = 0.0099 and <0.0001 respectively) and NF175 ( p = 0.0013 and <0.0001 respectively) compared to NF54. The percentage of Z2/hGK+/hGS- hepatocytes infected with parasites were not significantly different between the strains. For NF135 (Z1/hGK++/hGS- vs Z2/hGK+/hGS- = 0.0099; Z1/hGK++/hGS- vs Z3/hGK-/hGS+ and Z2/hGK+/hGS- vs Z3/hGK-/hGS+ <0.0001) and NF175 (Z1/hGK++/hGS- vs Z2/hGK+/hGS- = 0.0013; Z1/hGK++/hGS- vs Z3/hGK-/hGS+ and Z2/hGK+/hGS- vs Z3/hGK-/hGS+ <0.0001), there are significant differences between the percentage of infected hepatocytes within the different zones. There is no difference in the percentage of infected hepatocytes in the different zones for NF54. Data information: * p <0.05; ** p < 0.005; *** p <0.001; **** p <0.0001 F) Size of schizonts in Z1/hGK++/hGS-, Z2/hGK+/hGS- and Z3/hGK-/hGS+ hepatocytes on day 3 post invasion for the different Pf strains. For each parasite line, at least 100 schizonts were measured from each biological replicate. The median values +/- s.d. (as the sizes within the population is not normally distributed) from three biological replicates were used in a RM ANOVA test followed by a Tukey's multiple comparison test. Data information: * p <0.05; ** p < 0.005; *** p <0.001; **** p <0.0001 G) Percentage (+/- s.d.) of fPHH with NF54, NF135, and NF175 schizonts on day 5 p.i., under different parasite to hepatocyte ratios (starting from 8 sporozoites to 1 hepatocyte to 1 sporozoites to 8 hepatocytes). Two biological replicates were performed with two technical replicates in each. H) Total number of hepatocytes per well (+/- s.d.) for NF54, NF135 and NF175 on day 5 p.i., under different parasite to hepatocyte ratios (starting from 8 sporozoites to 1 hepatocyte to 1 sporozoites to 8 hepatocytes). Two biological replicates were performed with two technical replicates in each.