Fig. 3: PDCD6 deficiency promotes LAP.
From: PDCD6 regulates lactate metabolism to modulate LC3-associated phagocytosis and antibacterial defense
a, b Transmission electron micrographs of bacteria-containing single-membrane phagosomes (a) and quantification of cytosolic bacteria in bone marrow-derived macrophages (BMDMs) derived from Pdcd6fl/fl and Pdcd6Δmye mice (50 cells per group) (b) infected with L. monocytogenes. Scale bar, 0.5 µm. c, d Confocal microscopic images (c) and quantification (d) of the colocalization between L. monocytogenes (red) and LC3B puncta (green) in Pdcd6fl/fl and Pdcd6Δmye BMDMs (30 cells per group) challenged with RFP-L. monocytogenes. Scale bar, 2 µm. e Immunoblotting of LAP-associated molecules in phagosomes (left) and whole-cell lysates (right) isolated from Pdcd6fl/fl and Pdcd6Δmye BMDMs after bacterial challenge. f–i Flow cytometric analysis of the LC3B positivity ratio in Pdcd6fl/fl and Pdcd6Δmye BMDMs (f, g) or peritoneal macrophages (h, i) infected with L. monocytogenes (MOI: 5) (f, h) or S. Typhimurium (MOI: 5) (g, i) for the indicated periods. j, k Confocal microscopic images (j) and quantification (k) of the colocalization between L. monocytogenes (red) and LC3B puncta (green) in siCtrl and siPDCD6 THP-1 cells. Scale bar, 2 µm. l Flow cytometric analysis of the LC3B positivity ratio in siCtrl and siPDCD6 THP-1 cells infected with L. monocytogenes (MOI: 5) for the indicated periods. m–o Determination of reactive oxygen species (ROS) levels using DCFH-DA in the indicated BMDMs stimulated with L. monocytogenes. (m) Time-lapse confocal microscopic images (n) and quantification (o) of ROS fluorescence in phagosomes containing DCFH-SE-labeled L. monocytogenes (green). n Time (minutes) is indicated at the top of each panel. Scale bar, 5 µm. The averages of n = 16 (b), n = 10 (d, k), n = 3 (f–i, l, o), and n = 4 (m) biologically independent samples are shown. Data are shown as the mean ± SEM. Statistical significance in (b, d, f–i, k–m, o) was determined using the t test (and nonparametric tests). The data presented in (a, c, e–j, l–o) are representative of three independent experiments.
