Supplementary Methods v1MS 01-11-2016.docx
Supplementary Methods
Cell lines and cell culture:
The SUM159 cell line (ATCC, Manassas, VA) was maintained in Ham's F12 medium supplemented with 5% FBS, 5 µg/ml insulin, 1 µg/ml hydrocortisone, 100 U/ml antibiotic/antimitotic penicillin, and 100 mg/ml streptomycin. Cell lines were tested for mycoplasma by PCR and validated by short tandem repeat (STR) DNA fingerprinting using the AmpFLSTR Identifiler PCR Amplification Kit (cat 4322288, Life Technologies, Foster City, CA), according to manufacturer instructions.
Reverse phase protein array (RPPA)
Cells were plated in 60 mm plates and treated with or without doxycycline for 4 days. Cells were then washed twice in ice-cold PBS and lysed in 200 µl of RPPA lysis buffer (1% Triton X-100, 50 mM HEPES, pH 7.4, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 100 mM NaF, 10 mM Na pyrophosphate, 1 mM Na3VO4, 10% glycerol, and freshly made protease and phosphatase inhibitor) for 30 minutes on ice. Cell lysates were then centrifuged for 10 minutes at 14,000 rpm, and the supernatants were collected. Protein concentration was measured by bi-cinchoninic acid (BCA) assay and adjusted to 1 mg/ml. Samples were then treated with 4 × SDS sample buffer (40% Glycerol, 8% SDS, 0.25M Tris-HCL, pH 6.8, with 2-mercaptoethanol at 1/10 of the volume added before use) and boiled at 95ºC for 5 min, before being submitted to the RPPA core facility at The University of Texas M.D. Anderson Cancer Center for analysis. Cell lysates were then two-fold-serial diluted for 5 dilutions (from undiluted to 1:16 dilution) and arrayed on nitrocellulose-coated slide in 11x11 formats. Samples were probed with antibodies by CSA amplification approach and visualized by DAB colorimetric reaction. Slides were scanned on a flatbed scanner to produce 16-bit tiff image. Spots from tiff images were identified and the density was quantified by MicroVigene. Relative protein levels for each sample were determined by interpolation of each dilution curves from the "standard curve" (supercurve) of the slide (antibody). Supercurve is constructed by a script in R written by Bioinformatics. These Log2 value then median-centered for hierarchical cluster analysis includes all the detailed information pertaining to the antibodies used. The heatmap included was generated in Cluster 3.0 (http://www.eisenlab.org/eisen) as a hierarchical cluster using Pearson Correlation and a center metric. The resulting heatmap was visualized in Treeview.
Mouse Experiments
Genetically modified MDA-MB-231 cells were injected into the mammary fat pads of nude mice (1.5 × 106 per animal in 100 μl PBS) and measured at the indicated time points. Genetically modified SUM-159 cells were injected into the mammary fat pads of SCID mice (8.0 x106 per animal in 100 μl PBS) and measured at the indicated time points. After tumors developed and reached the size of 40-50 mm3, mice were randomized to receive doxycycline-containing (200 µg/ml) or doxycycline-free 30% sucrose-water to induce DUSP4 expression. Tumor sizes were measured twice weekly with digital calipers. Volumes were calculated using the formula v = (width2 × length)/2. Tumor growth rates of different groups were calculated and statistically analyzed. Mice were sacrificed when tumors reached 1500 mm3. The paired two-tailed t-test was used to determine the statistical significance of differences in DUSP4 expression between: 1) tumor datasets; and 2) groups of anchorage-independent growth. In vivo tumor growth was approximately exponential but exhibited variation between animals. Individual growth rates were estimated by linear regression of log-transformed tumor volumes over time and then compared using Student’s t-test to compare tumor growth rates between doxycycline-treated and doxycycline-untreated animals. Growth rates were summarized by mean values and 95% confidence intervals (CIs).