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Fig. 5 | Cancer Cell International

Fig. 5

From: A novel non-sulphamoylated 2-methoxyestradiol derivative causes detachment of breast cancer cells by rapid disassembly of focal adhesions

Fig. 5

EE-15-one exposure results in the disassembly of the focal adhesions, severance of actin stress fibres connected to focal adhesions, and loss of FAK phosphorylation. a MCF-7 cells were treated with 5 µM EE-15-one for 2 h before cells were processed for confocal microscopy. Cells were stained for F-actin (red) and FAK (green) using TexasRed conjugated phalloidin and FAK specific antibodies and FITC labelled secondary antibodies. Cells were imaged using a Zeiss LSM510 confocal microscope with a 63× mag objective, Scale bar, 20 µm. b For timelapse imaging, MCF-7 cells were transfected with LifeAct-mRFP (actin, red) and paxillin-GFP (focal adhesions, green). The following day cells were prepared for imaging every 30 s on a Zeiss LSM800 confocal microscope with environmental control. Cells were incubated with 5 µM EE-15-one. Scale bar, 20 µm. c MCF-7 cells were treated with 5 µM EE-15-one for the indicated timepoints before being processed for western blot. The top panel shows a representative western blot for phosphorylated FAK, total FAK and β-tubulin as loading control. The graph represents the average ratio of phosphorylated FAK over total FAK with both phosphorylated FAK and total FAK having been normalised to β-tubulin expression. Error bars represent SD and stars denote statistically significant differences between control and treated cells as calculated by student’s t-test with P < 0.05

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