1. Volume 3, Issue 3, Pages 630-637 (March 2013)
Suppression of Host p53 Is Critical for Plasmodium Liver-Stage Infection 
Alexis Kaushansky, Albert S. Ye, Laura S. Austin, Sebastian A. Mikolajczak, Ashley M. Vaughan, Nelly Camargo, Peter G. Metzger, Alyse N. Douglass, Gavin MacBeath, Stefan H.I. Kappe 
Cell Reports 
Volume 3, Issue 3, Pages (March 2013)
DOI: /j.celrep
Copyright © 2013 The Authors Terms and Conditions

2. Cell Reports 2013 3, 630-637DOI: (10.1016/j.celrep.2013.02.010)
Copyright © 2013 The Authors Terms and Conditions

3. Figure 1
The Use of Protein Microarrays to Study Host Signaling during Liver-Stage Malaria Infection
(A) Schematic representing steps required to obtain lysate microarray data from HepG2/CD81 cells either infected or uninfected with P. yoelii-GFP liver stages. Liver stages were allowed to develop for 24 hr. Abs, antibodies.
(B) Representative array images from three antibodies: total p53, LC3A, and p-Akt1/2/3 (pS473).
(C) Graph representing the ratio of infected (GFP-positive) cells to uninfected (GFP-negative) host cells for signals obtained for 46 separate antibodies, plotted against the log of the p value obtained. Each point represents a single antibody. Significant differences that pass multiple hypothesis testing (Holm-Bonferroni method) are shown in blue; nonsignificant differences are shown in red.
See also Figure S1.
Cell Reports 2013 3, DOI: ( /j.celrep )
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4. Figure 2
Key Host Signaling Pathways in Plasmodium Infected Hepatocytes
(A) Schematic showing the connectivity between host proteins significantly perturbed in liver-stage-infected cells as measured by protein lysate microarrays. Proteins for which total level or posttranslational modifications are increased in parasitized cells are shown in green, decreased levels in red. Select antibodies tested demonstrating a linear relationship between immunoblotting (western blotting [WB]) and lysate array (LA) are colored orange. Proteins for which transcripts where tested and changed are colored yellow.
(B) Demonstration that total p53 antibody (#9282) produces a linear relationship for data obtained using western blotting and lysate array. Data were fit using a linear regression.
(C) Quantitative PCR showing that transcript levels of Bad are decreased in infected hepatocytes.
(D) Transcript levels of Bcl-2 are elevated in infected hepatocytes.
Error bars represent SD of analytical replicates. See also Figure S1.
Cell Reports 2013 3, DOI: ( /j.celrep )
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5. Figure 3
Transgenic Mice and Pharmacological Perturbations Demonstrate a Critical Role for Host p53 in Liver-Stage Infection
(A) Mice with an additional copy of p53 (super-p53 mice) (n = 13), without p53 (p53 KO mice) (n = 7), or wild-type C57BL/6 mice (n = 20) were infected with 100,000 P. yoelii sporozoites. Liver-stage burden was monitored 42–44 hr after infection using quantitative reverse-transcription PCR. Parasite burden was significantly reduced in super-p53 mice (p = 0.004) and significantly elevated in p53 KO mice (p = 0.04).
(B–D) p53 levels increase in response to 48 hr Nutlin-3 treatment, as demonstrated by western blot using an anti-p53 antibody (B). An anti-α-tubulin antibody was used as a loading control. Nutlin-3 treatment (20 μM) 24 hr before and during infection (pre and post) dramatically reduces liver-stage burden of P. yoelii (C, middle) and P. berghei (D, middle). When treatment is applied beginning at time of infection and continuing until 24 hr after infection (post), liver-stage burden is also reduced, albeit less substantially for both P. yoelii (C, right) and P. berghei parasites (D, right). All liver stages were quantified 24 hr postinfection in HepG2/CD81 cells. Error bars represent SD between biological replicates.
(E) To monitor the effects of Nutlin-3 on P. yoelii (Py) sporozoite infection of HepG2/CD81 cells, cells were trypsinized 90 min postinfection, fixed, stained with an antibody to CSP, and subjected to flow cytometric analysis. Wells that were not infected with sporozoites were used as a control. Error bars represent SD of biological replicates.
(F and G) Nutlin-3 treatment dramatically reduces liver-stage burden in mice. A total of 50 mg/kg Nutlin-3 was administered once daily for 2 days to BALB/cJ mice. At the time of the last administration of Nutlin-3, mice were infected with 50,000 P. yoelii sporozoites. Livers were removed at 24 hr (F) or 44 hr (G) postinfection, and parasite 18S ribosomal RNA was assessed by quantitative real-time PCR. Signal was normalized to mouse GAPDH. For in vivo experiments, the mean is represented by a horizontal line, and the level of Py18S/GAPDH is shown for each individual mouse.
See alsoFigure S2.
Cell Reports 2013 3, DOI: ( /j.celrep )
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6. Figure S1
Biological Reproducibility and Linearity between Lysate Arrays and Western Blotting, Related to Figures 1 and 2
(A and B) Data obtained from 2 biological replicates of infected and uninfected cells on protein lysate microarrays. Signal from antibodies that recognize total p53 (A) or Bad pS112 (B) are shown.
(C) Graph representing the ratio of infected (GFP-positive) cells to uninfected (GFP-negative) cells for signals obtained, plotted against the log of the p-value obtained. Each point represents a single antibody of the Akt/mTor pathway. Only antibodies which demonstrate significant differences in signal between infected and uninfected cells are represented.
(D) Graph representing the ratio of infected (GFP-positive) cells to uninfected (GFP-negative) cells for signals obtained, plotted against the log of the p-value obtained. Each point represents a single antibody against total or phosphorylated p53. Only antibodies which demonstrate significant differences in signal between infected and uninfected cells are represented.
(E) Graph representing the ratio of infected (GFP-positive) cells to uninfected (GFP-negative) cells for signals obtained, plotted against the log of the p-value obtained. Each point represents a single antibody of the Bcl-2 family. Only antibodies which demonstrate significant differences in signal between infected and uninfected cells are represented.
Cell Reports 2013 3, DOI: ( /j.celrep )
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7. Figure S2
Nutlin-3 Targets Host Hepatocyte to Remove Liver-Stage Parasites, Related to Figure 3
(A) Asexual P. falciparum parasites diluted to 0.5% parasitemia, synchronized to > 95% ring stages then cultured with DMSO or 20μM Nutlin-3 for 48 hr. Parasitemia of treated and untreated cultures is similar.
(B) HepG2/CD81 cells were treated with Nutlin-3 or DMSO alone for 24h then infected with P. yoelii sporozoites. Liver stages were visualized by immunofluorescence and counted manually.
(C) HepG2/CD81 cells were treated with either DMSO or Nutlin-3 for 24 then incubated with P. yoelii sporozoites. Sporozoite traversal rates were monitored by addition of 1mM FITC dextran during incubation. Cells, which suffer wounded membranes take-up the FITC dextran and can be quantitated by FACS. Sporozoite traversal rates were similar between Nutlin-3 and DMSO-treated preparations, suggesting that Nutlin-3 does not inhibit cell traversal activity of sporozoites.
(D) Cells were treated and infected with sporozoites as described in 4B. Rather than total number of liver stages, the percentage of hepatocytes infected with sporozoites was monitored after 48 hr of Nutlin-3 treatment. A significantly lower percentage of hepatocytes are infected with sporozoites at this time point, suggesting Nutlin-3 treatment does not remove parasites by non-specific killing of hepatocytes.
(E) A lower percentage of sporozoites are found inside hepatocytes after Nutlin-3 treatment than in control-treated cells 90 min after invasion.
(F) Nutlin-3 treatment increases p21 in vivo. Mice were treated with Nutlin-3 daily for two days. 24 or 44 hr after the final treatment, RNA was monitored for p21 and GAPDH transcript levels by qPCR. Error bars represent standard deviation of analytical replicates.
(G) Nutlin-3 decreases size of Plasmodium liver stages that remain after treatment. Representative image of liver stage size in untreated (left) or Nutlin-3 treatment (middle) HepG2/CD81 cells after 24h of Nutlin-3 treatment. PVM marker UIS4 is shown in red. Liver stages in Nutlin-3 treated cultures are approximately half the size of untreated liver stages (right).
Cell Reports 2013 3, DOI: ( /j.celrep )
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