Figure 2.

Modulation of Host AMPK Activity Alters P. berghei Development

(A) Timeline of RNAi knockdown (KD) and infection.

(B) pAMPKα^T172 and pACC^S79 status in lysates of Huh7 cells 48 hr after AMPK α1 and α2 KD. Representative blot of three independent experiments (KD efficiency, mean±SEM, 64.3% ± 10.3%).

(C and D) Quantification of parasite size in AMPKα1/α2-depleted Huh7 cells (C) or AMPKα1^−/−α2^−/− MEFs (D), assessed by microscopy at 48 hr post-infection. Parasite size is the area defined by staining with the parasite membrane marker PbUIS4, as shown in the representative images. Nuclei were stained with Hoechst. More than 100 parasites were imaged and analyzed for each of the three independent experiments. ctrl, control; wild type (WT). Scale bars, 20 μm. ^∗∗∗∗p < 0.0001. The outliers in the boxplots represent 5% of data points.

(E) Timeline of transfection with AMPKα1-carrying plasmids and infection.

(F) Representative western blot of pAMPKα^T172 and pACC^S79 in lysates of Huh7 cells transfected with the truncated AMPKα1, constitutively active (CA), and mutated AMPKα1 (T172A) plasmids (see schematic of AMPKα1 domains and GST-tagged constructs in Figure S2A). GST was probed to detect transgenes.

(G and H) Representative images (G) and quantification (H) of parasite size in cells expressing AMPKα1-CA, AMPKα1-T172A, or GST only (empty plasmid) in transfected or untransfected cells (ctrl). Transfected cells were identified with anti-GST antibodies, and parasites were detected with anti-PbUIS4. Nuclei were stained with Hoechst. Parasite size distribution in GST-negative cells is shown in Figure S2B. A representative of three independent experiments is shown (30–60 parasites examined per condition). The outliers in the boxplot represent 10% of data points. Scale bars, 20 μm. ^∗∗p < 0.01; ns, non-significant.