Fig. 6. TRPM7 localizes proximal to the nascent phagosome.

a ImageStream-based analysis of protein localization in RAW 264.7 cells during phagocytosis of apoptotic cells is schematized. Similarity score measures colocalization of expression construct with CellTrace Violet-labelled cargo, and the score increases with increased colocalization. b ImageStream-based measurement of TRPM7 localization with phagocytosed apoptotic cells in fixed RAW 264.7 cells. Either FLAG-TRPM7 or GFP was transfected 16 h prior to incubation with apoptotic cells. For controls, the cells were subjected to 15 min pretreatment with Cytochalasin D (Cyto D; 1 μM) or Bafilomycin A1 (BafA1; 500 nM). After phagocytosis, the RAW 264.7 cells were fixed and immunostained for FLAG immunoepitope. Fluorescence of GFP and anti-FLAG was measured via ImageStream flow cytometry. Bar charts represent mean of n = 3 independent samples and the individual data points are overlayed; error bars are SEM. Representative cells are shown at right. Representative results of two independent experiments are shown. *p = 0.035. See also Supplementary Fig. 4a–c. c Confocal immunofluorescence microscopy of FLAG-TRPM7-expressing LR73 phagocytes after addition of 4 μm beads for phagocytosis. Fixed cells were immunostained for FLAG (TRPM7-green) and fluorescent beads (red). Top left: Brightfield shown merged with FLAG (magenta) and nuclei (blue); scale bar = 10 μm. Top right: merged ROI of FLAG and ER marker (PDI). Bottom left [c1]: FLAG-TRPM7 and PDI light are shown with merged pseudocolored image; scale bar = 3 μm. Bottom right: Single-channel images shown at bottom left. White dash box indicates ROI, and yellow dashed circled shows bead-containing phagosome. Single optical sections (0.5 μm) are shown. Additional single-channel images shown in Supplementary Figs. 4d, e and 5a. Source data are provided as a Source Data file, and statistical testing is described in “Statistics and Reproducibility”.