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. 2013 Sep 25;8(9):e75321. doi: 10.1371/journal.pone.0075321

Figure 4. Circumsporozoite protein does not affect basal or inducible expression of MHC class I.

Figure 4

(A) Expression of the full length (CSPf.l.) or mature “short” (CSPsh) form of the P. falciparum 3D7 CSP protein was detected by western blotting 24 hrs after transfection of HC-04 cells. (B) Cellular distribution of full length CSP 24 hrs post-transfection was visualized with a CSP-specific antibody (green) and immunofluorescence microscopy. Nuclei were stained with DAPI (blue). Arrows indicate transfected cells. (C) Real-time PCR analysis of MHC class I heavy chain, β2-microglobulin and TAP1 gene expression in cells transiently expressing of P. falciparum CSP. HC-04 cells transfected with the control vector plasmid or plasmid encoding either full length or short CSP were treated with AS (10% v/v) 4 hrs post transfection or left untreated. Transfected cells were isolated 24 hrs later using surface expression of mouse CD8α as a marker. Mean ± SD of the assay triplicates. All p* <0.0002 and indicated differences between control and AS-treated samples. (D) Percentages of cells transiently expressing CSP were identified by CD8α co-expression (upper panels) and MHC class I was assessed by flow cytometry (lower panel, light gray histograms - isotype control, dark gray histogram - MHC class I specific antibody). A proportion of cells exposed to either AS (10% v/v) or a mixture of recombinant TNFα and IFNγ at 24 hrs post transfection was further assessed for MHC class I expression at 48 hrs (light gray histograms - isotype control antibody, dark gray histogram - MHC class I in untreated cultures, solid line histogram – cultures treated with recombinant cytokines, dotted line histogram – cultures treated with AS). Data from one representative experiment. (E) The means ± SD of MFI for specific MHC class I staining obtained in 3 independent experiments. All p* <0.0001 and indicated differences between control and treated samples. (F) Expression of MHC class I heavy chain (HC) in total cell lysates of HC-04 cells transfected with CSP-expressing plasmids was assessed by western blot. Treatment with AS was done as described for D.