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. 2019 Mar 14;9:4490. doi: 10.1038/s41598-019-41016-4

Figure 2.

Figure 2

Androgen-triggered cytoskeleton changes, migration and invasiveness in MDA-MB231 and MDA-MB453 cells. In (a), quiescent MDA-MB231 cells were plated on coverslips and then left un-stimulated or stimulated for 20 min with 10 nM R1881. Thereafter, the cells were fixed, stained for F-actin using Texas-red conjugated phalloidin and analyzed by IF. Images representative of three independent experiments were captured and shown. The arrows indicate protrusions and ruffles in androgen-treated cells. Bar, 5μm. In (b), quiescent MDA-MB231 cells at sub-confluence were wounded and then left unstimulated or stimulated with R1881 (at 10 nM) for the indicated times. Cytosine arabinoside was included at 50 μM (final concentration) in the cell medium to avoid cell proliferation. About 14% of MDA-MB231 cells incorporated BrdU under quiescence condition. Challenging with 10 nM R1881 increased to 34% the number of cells incorporating BrdU, while simultaneous treatment with cytosine arabinoside reverted to 16% such number. When indicated, bicalutamide (Bic; at 1μM final concentration) and the S1 peptide (at 10 nM final concentration) were added 30 min before hormone stimulation. Contrast-phase images are representative of 3 different experiments, each in duplicate. Inset in (b), the wound area was measured using Image J Software and data are presented as % in wound width over the control, untreated cells (0 time). Data from 3 different experiments were collected and analyzed. Means and SEMs are shown. n represents the number of experiments. Quiescent MDA-MB231 (c) and MDA-MB453 (d) cells were used for invasion assay in Boyden’s chambers pre-coated with growth factor-reduced and phenol red-free Matrigel. The indicated compounds were added to the upper and the lower chambers. R1881 was used at 10 nM and bicalutamide (Bic) at 1μM. The S1 peptide was added (at 10 nM) 30 min before the hormonal stimulation. Here again, cytosine arabinoside (at 50 μM) was included in cell medium. About 12% of MDA-MB453 cells incorporated BrdU under quiescent, basal conditions. Challenging with 10 nM R1881 increased to 29% the number of MDA-MB453 cells incorporating BrdU, while simultaneous treatment with cytosine arabinoside reverted to 13% such number. After 24 h (for MDA-MB231 cells) or 36 h (for MDA-MB453 cells), invading cells from at least 30 fields /each membrane were counted as reported in Methods, using a DMBL (Leica) fluorescent microscope, equipped with HCPL Fluotar 20x objective. Results from three different experiments were collected and expressed as fold increase. Means and SEMs are shown. n represents the number of experiments. *p < 0,05 for the indicated experimental points versus the corresponding untreated control.