Supplementary Materials Editorial Process TRA-19-273-s001. for 24 hours. Fluorescent and DIC images of a representative cell from Mouse monoclonal to SRA at least 16 cells and 2 impartial experiments are shown. Scale bar = 5 m. Physique S3. Analysis of pH in the phagosome and associated vacuole using pH\sensitive and insensitive dye conjugated dextran. J774A.1 macrophages preloaded with dextran were co\incubated with MDA\MB\453 malignancy cells opsonized with trastuzumab as explained in the legend for Fig. ?Fig.3E.3E. Fluorescent and DIC images are shown, with a collection drawn across the phagosome/vacuole. Line intensity plot represents the normalized intensity for the two fluorescent signals GNE-7915 novel inhibtior (Alexa 488 and pHrodo Red, shown in blue and reddish, respectively) detected along the yellow line. GNE-7915 novel inhibtior Data for each fluorophore are normalized against the maximum transmission level. The ratio of fluorescent intensities in the phagosomes and vacuoles were quantitated and 40% (= 110) of vacuoles were found to have similar pHrodo Red:Alexa 488 intensity ratios in both the vacuole and adjacent phagosome. Yellow arrows indicate the location of the vacuole, and images of a representative cell from 110 cells and 3 impartial experiments are shown. Scale bar = 5 m. Physique S4. Phagosome\associated vacuoles are observed with multiple effector and target cell types. A, MDA\MB\453 cells opsonized with Alexa 488\labeled trastuzumab (pseudocolored reddish) were co\incubated with mouse bone marrow\derived macrophages for 6 hours, followed by addition of Lysotracker Red (pseudocolored green) and imaging (fluorescence and DIC). B, MDA\MB\453 cells opsonized with Alexa 647\labeled trastuzumab (pseudocolored reddish) were co\incubated with human monocyte\derived macrophages for 6 hours and treated/analyzed in for panel A. C, Raji B cells opsonized with Alexa 647\labeled rituximab or SK\BR\3 cells opsonized with Alexa GNE-7915 novel inhibtior 488\labeled trastuzumab (pseudocolored reddish) were co\incubated with J774A.1 macrophages for 4.5 hours or 6 hours, respectively, and treated/analyzed as in panel A. Yellow arrows in A\C show the location of the vacuole. Images of representative cells from at least 34 cells and 2 impartial experiments are shown. Scale bars = 5 m. TRA-19-273-s002.docx (7.0M) GUID:?351AA8EA-D439-42E5-B38D-F1EC510D8EE8 Movie S1. Formation of the phagosome\associated vacuole. The movie corresponds to Figure ?Figure1A.1A. Time\lapse images of transmitted light (left), Alexa 647\labeled dextran preloaded in lysosomes of J774A.1 macrophages (center) and Alexa 555\labeled trastuzumab originating from opsonized MDA\MB\453 cells (right) are shown. Time around the upper left is shown in the hours:moments:seconds format. The movie plays at a speed of 1500 actual\time. Scale bar = 5 m. TRA-19-273-s003.mp4 (7.9M) GUID:?8D8A8128-1749-48AA-A1BB-9A4F9772E03C Movie S2. Redistribution of the contents of the phagosome and the associated vacuole. The movie corresponds to Figure ?Figure2B.2B. Time\lapse images of Alexa 488\labeled dextran preloaded in lysosomes of J774A.1 macrophages (upper left, pseudocolored reddish), Alexa 555\labeled dextran preloaded in lysosomes of MDA\MB\453 malignancy cells (lower left, pseudocolored green) and DIC (upper right) are shown. Time in the upper right panel is shown in the hours:moments format. The movie plays at a speed of 2600 actual\time. Scale bar = 5 m. TRA-19-273-s004.avi (9.1M) GUID:?94C0C3E2-B311-4572-860E-007639724B81 Abstract Despite the rapidly expanding use of antibody\based therapeutics to treat cancer, knowledge of the cellular processes following phagocytosis of antibody\opsonized tumor cells is limited. Here we statement the formation of a phagosome\associated vacuole that is observed in macrophages as these degradative compartments mature following phagocytosis of HER2\positive malignancy cells in the presence of the HER2\specific antibody, trastuzumab. We demonstrate that this vacuole is a distinct organelle that is closely apposed to the phagosome. Furthermore, the size of the phagosome\associated vacuole is increased by inhibition of the mTOR pathway. Collectively, the identification of this vacuolar compartment has implications for understanding the subcellular trafficking processes leading to the.