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to mediate the acquisition of lysosomal components in the absence of the PI3K p110a. Our findings advance the novel concept that other classes of PI3K in addition to Vps34 regulate phagosome maturation. that knockdown of p110a did not MedChemExpress Trametinib Impair phagocytosis of the prey involved in this study. Phagosomes from p110a Knockdown Cells have Less p110a and PIP3 but Similar Levels of PI3P We examined whether p110a is recruited to phagosomes and assessed phagosomal levels of its phosphoinositide product, PIP3. PMA-differentiated control and constitutive knockdown cells were fed BSA-coupled 3 mm magnetic beads and 4 hour phagosomes were isolated by magnetic pulldown. Phagosomes were then either fixed for staining with antibodies against PI3P and PIP3, or lysed for determination of p110a phagosomal recruitment by immunoblotting. Our results showed that the PI3K p110a was associated with phagosomes isolated from THP-1 cells, and that the class IA PI3K phophoinositide product, PIP3 was also present on these phagosomes. Phagosomes isolated from p110a knockdown cells displayed significantly less recruitment of this PI3K, and a decreased amount of PIP3, though levels of PI3P were unaffected. Flow organellometry of bead phagosomes indicated that PI3P levels appeared to be unchanged throughout phagosome maturation. Results Knockdown of the Class IA PI3K p110a does not Impair Phagocytosis of Prey Less than or Equal to 3 mm in Diameter Selective, stable knockdown of expression of the class IA PI3K p110a in the THP-1 human monocytic cell line was done by lentiviral transduction as described previously. It has been documented that inhibition of PI3K activity impairs phagocytic uptake of prey larger than 4 mm. To test the ability of constitutive p110a knockdown cells to take up prey less than or equal to 3 mm in diameter, we fed phorbol-12-myristate-13acetate -differentiated control and p110a knockdown cells BSA-coupled 3 mm magnetic beads. Alternatively, parallel cell cultures were infected with the minimally virulent, opportunistic organism M. smegmatis. Prey, both beads and bacteria, were covalently surface labelled with the fluorescent dye Alexafluor 633 succinimidyl ester to allow quantitation of uptake by flow cytometry. Both control and p110a constitutively deficient cells displayed equivalent levels of uptake, thereby indicating Flow Organellometry of Phagosomes from p110a Knockdown Cells Shows Similiar Levels of Rab5B and EEA-1, but a Defect in Acquisition of Lysosomal Marker Proteins Control and constitutive p110a knockdown cells were fed BSAcoupled 3 mm magnetic beads and phagosomes were isolated at various timepoints. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/2221058 Vacuoles were then stained for various phagosome maturation markers and analyzed by flow organellometry. p110a knockdown cells acquired similar levels of the early endosomal small GTPase, Rab5B, and early endosomal autoantigen 1 . However, acquisition of LAMP-1 and LAMP-2 by p110a deficient cells was markedly impaired. Nevertheless, both cell types were found to express similar levels of these markers at the whole cell level . Furthermore, confocal fluorescence microscopy showed no defects in biosynthetic sorting of LAMP-1 from 2 PI3K p110a and Phagosome Maturation 3 PI3K p110a and Phagosome Maturation the trans-Golgi network to the endosomal system. Thus, deficient acquisition of these lysosomal membrane proteins may have been the result of null fusion events between maturing phagosomes, endosomes and lysosomes. Our kinetic studies wh