Figure 3

HGF-induced ruffling. A: HeLa-cells were pre-treated either under Mock-conditions or with an siRNA directed against the mRNA of the ACTR3 component of the Arp2/3 complex. Later, HGF was added, where indicated. Red: Actin, Blue: Nuclei. Scale bar: 20 μm. B: After image segmentation and measurement by CellProfiler, a model was trained in CellClassifier, outlined images showing predictions were produced. Grey: actin, green: nuclei, red outlines: ruffling cells, blue outlines: non-ruffling cells, yellow outlines: mitotic cells. C: Part of the Excel-sheet generated by CellClassifier. D: Illustration of CellClassifier output, left: heat maps. Positions of mock treated wells +HGF: B02-E02, no HGF: B11-E11. siRNAs against ACTR3 mRNA were positioned at F03-F06 (+HGF) and F07-F11 (no HGF). Only wells of interest were imaged. E: Summary of the experiment generated outside CellClassifier. Cells were pre-treated with siRNA and incubated with HGF as indicated. Each bar shows the median and standard deviation of 4 wells. With ACTR3 each well was treated with a different siRNA against ACTR3, yielding virtually identical effects.