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03 août 19171917/08/03 (A46).Appartient à l’ensemble documentaire : PoitouCh

Enhanced phagocytosis of zymosan in Inpp4a-deficient cells.

<p>(A) Raw264.7 cells deficient in Inpp4a (seq1 and seq2) or control cells were incubated for 15 min with IgG-coated zymosan. (B) Peritoneal macrophages from wild type (WT) or Inpp4a knock out (KO) mice were incubated for 15 min with IgG-coated zymosan. (A, B) The number of fluorescent particles was counted in merged images, and the number of zymosan particles within the cells was calculated. The results from three separate experiments are shown as the means ± s.e.m. **P<0.01</p

Timing of recruitment of Inpp4a and PtdIns(3,4,5)P₃ during phagosome formation.

<p>(A, B) RAW264.7 cells were transfected with mCherry-Inpp4a along with EGFP-[PH1-PH2- PH1(MyoX)]. The cells were incubated with E-IgG at 37°C under a microscope. (B) The fluorescence intensity of each probe was quantified as described under "Materials and Methods" and shown as % of respective maximum values. The time course of the Inpp4a association is shown as a combined result from 12 different cells that were transfected with mCherry-Inpp4a (open circles), while that of PtdIns(3,4,5)P₃ is calculated from 3 cells that were transfected with both mCherry-Inpp4a and EGFP-[PH1-PH2-PH1(MyoX)]. Time zero in the figures indicates the time when the mCherry-Inpp4a fluorescence around E-IgG peaked.</p

Inositol Polyphosphate-4-Phosphatase Type I Negatively Regulates Phagocytosis via Dephosphorylation of Phagosomal PtdIns(3,4)P₂

<div><p>Phagocytosis is a highly conserved process whereby phagocytic cells engulf pathogens and apoptotic bodies. The present study focused on the role of inositol polyphosphate-4-phosphatase type I (Inpp4a) in phagocytosis. Raw264.7 cells that express shRNA against Inpp4a (shInpp4a cells) showed significantly increased phagocytic activity. The introduction of shRNA-resistant human Inpp4a abolished this increase. Macrophages from Inpp4a knockout mice showed similar increases in the phagocytic activity. Inpp4a was recruited to the phagosome membrane by a mechanism other than the direct interaction with Rab5. PtdIns(3,4)P₂ increased on the phagosome of shInpp4a cells, while PtdIns(3)P significantly decreased. The results indicate that Inpp4a negatively regulates the phagocytic activity of macrophages as a member of the sequential dephosphorylation system that metabolizes phagosomal PtdIns(3,4,5)P₃ to PtdIns(3)P.</p></div

Enhanced phagocytosis of E-IgG in Inpp4a-deficient cells.

<p>(A) Control (open circles) or shInpp4a cells (closed circles) were incubated for the indicated times with E-IgG. The numbers of E-IgG on the outer surface of the cells (binding) and within the cells (phagocytosis) were determined as described in the Methods section. (B) Control (cont) or shInpp4a (seq1) cells were incubated on ice for 30 min with E-IgG. The bound E-IgG was visualized by staining with Alexa488-anti-rabbit IgG, and was counted under a microscope. (C) Control (cont) or shInpp4a (seq1) cells were transfected with EGFP-tagged human Inpp4a, cultured for 24 h, and added with E-IgG. The phagocytosis of the EGFP-expressing cells and non-expressing cells was determined under a microscope. The results from three separate experiments are shown as the means ± s.e.m.</p

Preparation of Inpp4a-deficient cells.

<p>(A) Inpp4a mRNA was detected by RT-PCR with primer pairs shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142091#pone.0142091.s004" target="_blank">S1 Table</a>. (B) Total RNAs from the mouse heart and Raw264.7 were subjected to RT-PCR. (C) Two lines of shInpp4a cells (seq1 and seq2) were prepared with the respective target sequences. The Inpp4a protein level was analyzed by western blotting. (D) Peritoneal macrophages were prepared from wild type (WT) or Inpp4a knockout (KO) mice. The Inpp4a protein level was determined by western blotting.</p