All Mkl1 variants were expressed as C terminal RFP tagged fusions. Dorsomorphin chemical structure An empty vector expressing RFP alone was previously described. HC11 mammary epithelial cells, kindly provided by Dr. N. Hynes, were grown in RPMI 1640 medium supplemented with 10% FCS, 5 ug ml insu lin Inhibitors,Modulators,Libraries and 10 ng ml epidermal growth factor. In most of the experiments, the HC11 cells were starved in 0. 03% FCS RPMI without EGF. To obtain HC11 cells stably expressing FL Mkl1 RFP, mutB1 Mkl1 RFP, SAP Mkl1 RFP or RFP alone, Inhibitors,Modulators,Libraries cells were transfected using FuGENE 6 and selected with Geneticin for 14 days before fluorescence activated cell sorting of RFP positive cells on a Vantage SE. Cell viability of the four HC11 cell strains was assessed by the CellTiter Blue viability assay.
Cell proliferation assay Proliferation rates of the HC11 cell strains were determined Inhibitors,Modulators,Libraries using BrdU incorporation assay. After 24 h of star vation, cells were plated in triplicate on Black 96 well mi crotiter plates at 5 103 cells well in 3% FCS RPMI and allowed to pro liferate for 0, 24, 48, 72 and 96 h before labeling with BrdU for 2 h. BrdU incorporation into newly synthesized DNA was determined according to the manufacturers protocol using a Luminometer Mithras LB940. Experimental values were normalized to the values of HC11 SAP cells at the time point 0. Data represent means SD from three independ ent experiments. Cell migration assay Cell migration was assayed using transwell polycarbonate membrane inserts with 8 um pores as described. After 24 h of starvation, 5 104 cells were plated in the top in sert chamber with 100 ul serum free RPMI.
The lower chamber was filled with 600 ul 10% FCS RPMI. Cells were allowed to migrate across the filter for 22 h at 37 C before fixation and crystal violet staining. Images of duplicate in serts were acquired on a Nikon Eclipse E600 using 10 magnification and a color CCD camera. Migration was quantified by measuring the area covered by migrated cells using the Fiji distribution of ImageJ. Inhibitors,Modulators,Libraries Data represent means SD from three independent experiments. Mechanical stimulation of cells 2 105 HC11 cells well were seeded in BioFlex 6 well culture plates coated with either growth factor reduced Matrigel or fibronectin. Inhibitors,Modulators,Libraries Cultures were starved for 24 h before applying either equibiaxial cyclic strain or static strain at 37 C for 1 h using Flexcell FX 4000.
Cells cultured under the same conditions and not exposed to strain were used as a resting control. After mechanical stimulation, cells were lysed and total RNA was isolated using the RNeasy Mini Kit. Transcript profiling and bioinformatics selleck analysis HC11 cell strains stably expressing Mkl1 variants were starved for 48 h before total RNA was extracted, converted into labeled cDNA and hybridized to Affy metrix GeneChip Mouse Gene 1. 0 ST arrays.