Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Activation-induced thrombospondin-4 works with thrombospondin-1 to build cytotoxic supramolecular attack particles

doi: 10.1073/pnas.2413866122

Figure Lengend Snippet: TSP-4 and TSP-1 localize predominantly together to LGs. ( A ) Representative stimulated human CTL expressing TSP-1-Flag (cyan) and TSP-4-HA (magenta) forming an IS with the anti-CD3 coated coverslip. Cells were fixed and stained with anti-GzmB (yellow), anti-Flag, and anti-HA antibodies. TSP-1 and TSP-4 were acquired in STED microscopy mode while GzmB was acquired in confocal mode. Shown are the merge image of all three channels and the images of the individual channels. On the Right is depicted the brightfield image of the same cell. (Scale bar, 2 µm.) ( B ) Enlarged pictures of organelles, which are positive for GzmB, TSP-1-Flag, and/or TSP-4-HA extracted from four different human CTL images as displayed in ( A ). (Scale bar, 0.5 µm.) ( C and D ) Object-based colocalization analysis of all three proteins displayed as a scatter dot plot with the values of individual cells superimposed with a box plot comprising the median (line) and the mean value (stippled line). Panel ( C ) shows the % of organelles with or without TSP-1 and/or TSP-4 among all individual organelles and panel (D) shows the % of LGs, i.e. organelles that display GzmB labeling, with or without TSP-1 and or TSP-4. N donors = 2, n cells = 32, 1,427 organelles.

Article Snippet: TIRF and spinning disc confocal microscopy were carried out on Nikon ECLIPSE Ti2-E microscope equipped with a Yokogawa CSU-W1-SoRA; STED was carried out on Abberior instruments GmbH QUADScan; SIM on ultrathin sections on a Zeiss ELYRA PS.1 microscope; and dSTORM on an ONI Nanoimager microscope.

Techniques: Expressing, Staining, Microscopy, Labeling