8.0 m pet membrane

The cell invasion assay was performed using a 24 well trans-well invasion chamber with an 8.0 µm PET Membrane (Corning, Glendale, AZ, USA, Cat. #354480). Briefly, the invasion chamber was removed from −20 °C and allowed to come to room temperature. The invasion chamber was rehydrated in serum-free media for 2 h at 37 °C in a tissue culture incubator. The media (500 µL) containing 10% FBS was added to the lower compartment of the trans-well as a chemo-attractant. Cells were pre-treated with 10 μM gDEC for 3 Days. After 3 days, 50,000 cells were suspended in 500 µL of the media containing 1% FBS, 10 μM gDEC, and added to the upper compartment of the invasion chamber. The same number of cells without gDEC treatment served as a control. Following the incubation for 48 h at 37 °C in the tissue culture incubator, cells were fixed with ice-cold methanol at −20 °C for 10 min and stained with Coomassie brilliant blue. The remaining cells from the upper compartment were removed using cotton-tipped swabs with gentle swabbing.

Breast cancer MDA-MB231 cells were grown in DMEM medium (Corning, NY, Cat#10–013-CV) with 10% FBS and 100 IU/ml penicillin-streptomycin at 37°C and 5% CO₂ and then plated at a density of 1.32 × 10⁴ in 0.3 ml of DMEM medium without FBS into the upper chamber of a Bioboat insert fitted with a 8.0 µm PET membrane (Corning, NY, Cat#354578). For migration assays, the filter was uncoated. For invasion assays, it was pre-coated with 100 µl Matrigel (BD Biosciences, Cat#356234) at concentration of 272 µg /ml for 1–2 hr at room temperature. Medium containing 10% FBS (0.6 ml) was placed into the lower chamber as a chemo attractant. The compound (final concentration of 5 µM) or a matching amount of DMSO was added to both chambers. After 24 hr or 48 hr the cells were fixed with −20°C methanol for 15 min and then stained with Trypan blue for 5 min. Cells on the upper surface were removed using cotton swabs. Cells present on the underside of the membrane were photographed using an EVOS FL Cell Imaging System (Invitrogen, CA) and the images were thresholded for presentation and counting using Image J (National Institutes of Health, Bethesda, MD, SCR_003070). The assays involving MCF7 cells were identical except that they were performed 24 hr post transfection.