Plasmodium berghei-Mediated NRF2 Activation in Infected Hepatocytes Enhances Parasite Survival

Figure 3

p62 activates NRF2 by recruiting KEAP1 to the PVM. (a) Primary mouse hepatocytes were infected with PbmCherry parasites (red). 6 hpi, cells were fixed and stained with either anti-UIS4 (magenta; upper panel) or anti-LC3 antibodies (magenta; middle panel) to visualize the PVM. p62 was stained using an anti-p62 antibody or an antibody specifically recognizing p62 phosphorylated at serine 351. Images were taken at a confocal laser scanning microscope and deconvolved using Huygens Professional. Scale bar: 10 μm. Note that p62 localizes to the PVM and is phosphorylated at S351. (b) Quantification of (a). Graph shows Manders’ Overlap Coefficient (MOC) for p62 and UIS4, phosphorylated p62 and LC3B, and phosphorylated p62 and p62. MOC was calculated using FIJI. parasites per staining. Each dot represents one parasite with the pink dot representing the parasite shown in (a). (c) HuH7 WT (upper panel) and p62^-/- (second panel) cells stably expressing GFP-KEAP1 (green) were infected with P. berghei wildtype sporozoites (PbWT). 6 hpi, cells were fixed and stained with anti-UIS4 antibodies to visualize the PVM (magenta) and anti-p62 antibody (here in red). In addition, p62^-/- cells were transiently transfected with either mCherry-p62 (third panel; red) or mCherry-p62-T350A (lowest panel; red). Images were taken by confocal microscopy and deconvolved with Huygens Professional. Note that p62 T350 is required for KEAP1 recruitment to the PVM. Scale bar: 10 μm. (d) Quantification of (c). The percentage of UIS4-positive parasites showing GFP-KEAP1 associations with the PVM was determined in all four conditions. The graph depicts mean and SD of three independent experiments. -values were calculated using a one-way ANOVA test followed by Tukey’s post hoc test (ns: and ). per experiment for WT and p62^-/- cells. per experiment for the addback conditions. (e) Confirmation of p62 knockout by western blot. HuH7 WT cells, two p62^-/- clones, and the respective BFP-p62 addback cell lines were lysed, and western blot analysis was carried out. Whole protein lysates were run on a 12% SDS gel. p62 was detected using a mouse anti-p62 antibody. α-Tubulin was detected sequentially with a mouse anti-α-Tubulin antibody and used as a loading control. (f) HuH7 p62^-/- cells (clone G2) stably transduced with the OKD48 NRF2-reporter construct [28] were infected with PbmCherry sporozoites. 5 hours post infection (hpi) cells were sorted by FACS into an infected and a noninfected population. 6 hpi, luciferase activity was measured, and the signal was normalized to the number of living cells in each sample. As a positive control, cells treated with 1 μM sodium arsenite (ASN) for 6 hours were used. Sorted infected cells were compared to sorted noninfected cells, while ASN treated cells were compared to untreated cells. The graph shows the relative induction compared to the respective control. The experiment was carried out 3 times independently. Mean and SD are depicted. Significance was determined by -test (ns: and ).

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