Change in cell shape is required for matrix metalloproteinase-induced epithelial-mesenchymal transition of mammary epithelial cells Page: 5 of 26
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Materials and Methods
Cell culture and reagents. Use of SCp2 mouse mammary epithelial cells and treatment
with MMP3 were as previously described [Radisky et al., 2005], with a 3 day time point
for all experiments. The following reagents were used at the given concentrations: H202
(25 pM, Sigma); NAC (10 mM, Sigma); GM6001 (40 pM, Calbiochem). For
measurements of projected cell area, phase contrast images of individual cells were
outlined and processed with Image J software. Transfections were performed with
lipofectamine-2000 (Invtrogen) as previously published [Radisky et al., 2005]. For
luciferase assays, cells were transfected with plasmids expressing 3TP-luciferase and
CMV-renilla in a 10:1 ratio, and relative luciferase was assessed using the Dual-Glo
Luciferase Assay System (Promega) and a Veritas Microplate Luminometer (Turner
Biosystems). Plasmids for expression of YFP-Raclb, YFP-RaclN17, and CMV-Raclb
and methods for selective siRNA knockdown of Raclb and for assessing ROS using
DCFDA (Invitrogen) were described previously [Radisky et al., 2005]; pEYFP-C1
(Clontech) was used as a control for transfection experiments. Recombinant human TGF-
f 1 (240-B, R&D Systems) was used at 25 ng/ml final concencentration.
Quantitative real-time PCR analysis. Transcript levels were measured using RT/PCR by
isolating RNA (Tri-pure; Roche Diagnostics), synthesizing cDNA and performing
quantitative real-time PCR using ABI protocols and software (Lightcycler, Roche).
Expression levels were assessed using TaqMan assays (Roche): sm-actin,
Mm01546133_ml; GAPDH, Mm99999915_gi. We assessed Raclb using a custom assay
(forward primer, 5'- TGGACAAGAAGATTATGACAGATTGC-3'; reverse primer, 5'-
CCCTGGAGGGTCTATCTTTACCA-3'; probe, 5' CCGCAGACAGTTGGAGA-3').
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Nelson, Celeste M.; Khauv, Davitte; Bissell, Mina J. & Radisky, Derek C. Change in cell shape is required for matrix metalloproteinase-induced epithelial-mesenchymal transition of mammary epithelial cells, article, June 26, 2008; Berkeley, California. (https://digital.library.unt.edu/ark:/67531/metadc901999/m1/5/: accessed April 23, 2019), University of North Texas Libraries, Digital Library, https://digital.library.unt.edu; crediting UNT Libraries Government Documents Department.