Fig. 2

Sublethal doses of Dox induce pro-invasion/migration phenotype. A Brightfield images show changes in cell morphology with Dox treatment. Inset shows zoomed in view. B Cells were fixed and stained with Rhodamine-Phalloidin to visualize changes in the actin cytoskeleton and DAPI to visualize nuclei. Cells were imaged with a confocal microscope at × 63 magnification. C Cells were seeded in 24-well plate, and a scratch was introduced in each well prior to treatment. Random field-of-view of wound healing for each condition was shown at the time points indicated. D Quantification of wound area from C expressed as % area on day 0 (Mean ± SEM, *p < 0.05 vs. Veh. n = 3 in triplicate). E Cells were seeded in 6-well plates. At each time point, viable cell number was assessed by MTT assay (Mean ± SEM, ***p < 0.001 vs. Veh, n = 3). F Cells were treated for 24 h as shown prior to trypsinization and re-seeding in serum-free media into Boyden chambers with 0.8-μm pores, with 10% FBS media as chemoattractant. Migrated cells were stained with crystal violet and random brightfield images were taken with an EVOS microscope. Quantification of migrated cell numbers in F from 8 random fields-of-view for each condition. Data are fold change over Veh. (Mean ± SEM, ***p < 0.0005 vs. Veh n = 3). G qRT-PCR analysis of MMP-1, MMP-2, MMP-9, and MMP-14 was performed with actin as reference gene. Data are shown as mean normalized expression (Mean ± SEM, **p < 0.01 vs. veh, n = 3). H Cells were serum starved for 24 h post-treatment. Conditioned media was collected, and gelatin zymography assay was done to assess MMP-2 and MMP-9 activity. Picture shown is representative of n = 3. Quantification of gelatin zymography; relative intensity of bands in gel plotted (n = 3). I Cells were seeded in 100-mm dishes and serum starved for 16 h prior to treatment, followed by re-seeding in fibronectin-coated 12-well plates. After 1 h, the number of attached cells following multiple washes with PBS was estimated with MTT assay. Expressed as % attached cells of veh (Mean ± SEM, **p < 0.01 vs veh, n = 4). All treatments with Dox are at 0.4 μM unless otherwise noted