Extended Data Fig. 1: The mCherry-niche system in vitro.
From: Metastatic-niche labelling reveals parenchymal cells with stem features
a, sLP–mCherry design. b, Fluorescence images of labelling-4T1 cells after thawing. Scale bar, 10 μm. c, Representative FACS plot of labelling-4T1 cells. d, In vitro cultures of the indicated cell types with LCM: culture scheme and representative fluorescence images of HC11 (mouse mammary epithelial cells) and hNLF (human normal lung fibroblasts) with LCM (scale bar, 10 μm). e, FACS plots of 4T1, HC11, RAW264.7 (mouse macrophages), hNLF and mouse breast CAFs cultured with LCM. f, FACS analysis of 293T cells cultured with LCM, at different time points after LCM removal (black dots); white dots show the theoretical decrease considering the cell proliferation rate only (the amount of 293T cells labelled with mCherry after 24 h incubation with LCM was set to 100%). g, Representative fluorescence image of 4T1-CD63–GFP cells cultured with LCM. Scale bars: main panels, 5 μm; enlarged region, 1 μm. h, Representative correlative light and electron microscopy of labelling-4T1 cells showing re-uptake of sLP–mCherry (n = 5 different cells analysed). Top left, bright-field image overlaid with mCherry immunofluorescence (∼700 nm optical section). Bottom left, electron microscopy of the same cell (∼70-nm section thickness). Centre, best approximation of immunofluorescence–bright-field–electron microscopy overlay (scale bar, 5 μm). Right, electron microscopy of the outlined regions (centre, labelled a–c) (black arrows point at vesicular structures containing mCherry; scale bar, 1 μm). i, j, Analysis of in vitro labelling potential of soluble fraction and extracellular vesicles isolated from LCM by FACS. i, Schematic representation of LCM fractionation. j, HC11 cells cultured with either LCM, soluble fraction after depletion of extracellular vesicles (soluble) or purified extracellular vesicles. k, ImageStream analysis of mCherry+ extracellular vesicles in LCM (16% of total extracellular vesicles are mCherry+). Data are representative of three (b), ten (c) or two (d–g, j, k) independent experiments.
