MAP4K4 promotes host cell motility and TNFα-induced invasiveness.

Abstract

<p><b>A</b>) Confocal IFA of intracellular localization of MAP4K4 in parasitized (upper) and BW720c-cured (lower) TaC12 cells. Red: actin, green: MAP4K4, blue: TaSP. Right: magnification of TaC12 lamellipodium showing accumulation of MAP4K4 at leading edge. Schema shows localization of MAP4K4 at leading edge of cell. <b>B</b>) IB analysis of untreated and BW720c-treated TaC12 cells using anti-MAP4K4, anti-ERM, anti-Hck and anti-tubulin antibodies. Bar diagram below shows mean protein abundances −/+ SDs. in control and BW720c-treated TaC12 cells. <b>C</b>) <i>In vitro</i> kinase assay comparing MAP4K4 immunoprecipitated either from infected or cured TaC12 cells. Upper: IB of immunoprecipitated MAP4K4, lower: autorad of total protein phosphorylation activities immunoprecipitated. <b>D</b>) Upper: IB analysis with anti-MAP4K4 and anti-tubulin antibodies using lysates of TaC12 cells previously incubated with increasing concentrations of conditioned medium. Lower: Mean integrated densities of MAP4K4 bands (−/+ SDs, normalized to tubulin) <b>E</b>) Quantification of MAP4K4 depletion by qRT-PCR using primers p81 and p79 (upper, see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004003#ppat-1004003-g005" target="_blank">Fig. 5A</a>) or IB (lower) 24 h after siControl or siMAP4K4 transfection. <b>F</b>) 12 h single cell migration analysis of cells transfected with either siControl or siMAP4K4_1 or siMAP4K4_2. Box plot show means of path lengths (n = 34, 38, 44). <b>G</b>) Matrigel invasion assay: Bright field microscopy images (100× magnification) of transmigrated siControl or siMAP4K4_1 or siMAP4K4_2 transfected cells after 24 h in the presence of 25 ng/ml TNFα. <b>H</b>) Quantification of images shown in G expressed as relative transmigration compared to siControl and SDs. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004003#ppat.1004003.s003" target="_blank">Figures S3</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004003#ppat.1004003.s004" target="_blank">S4</a>.</p