Additional file 3: of Pyruvate carboxylase supports the pulmonary tropism of metastatic breast cancer

Figure S3. Transient induction of EMT does not induce expression of PC. (A) Photomicrographs showing EMT-like changes in cellular morphology of the control (scram) and PC-depleted (shPC25 and shPC28) 4 T1 cells upon treatment with exogenous TGF-β1 (5 ng/ml) for 4 days. (B) Immunoblot analyses for PC in cells shown in Panel A. β-tubulin served as a loading control. (C) The RAS transformed MCF-10A-T1 k cells were constructed to express a doxycycline-inducible vector encoding the EMT transcription factor Snail. Expression of PC was analyzed upon addition of doxycycline (Dox) at the indicated concentrations. Expression of Snail and β-tubulin (β-Tub) served as loading controls. (PDF 1401 kb

Additional file 1: of Pyruvate carboxylase supports the pulmonary tropism of metastatic breast cancer

Figure S1. PC expression is not correlated with primary tumor size. Patient samples within the METABRIC dataset were analyzed for PC expression in relation to primary tumor size. Data are analyzed by a nonparametric Spearman correlation resulting in the indicated r and P values. The linear regression for these data is also shown. (PDF 235 kb

Additional file 4: of Pyruvate carboxylase supports the pulmonary tropism of metastatic breast cancer

Figure S4. PC is not required for cell migration. (A) Control (shScram) and PC depleted (shPC28) cells were plated into 8 μm transwell inserts in serum-free media containing 1 or 4 mM pyruvate. Cell migration was quantified 12 h after plating using calcein AM. SFM represents wells where the bottom chamber was filled with serum-free media as negative control. Values are presented as mean relative fluorescence units, ±SEM. (B) Control (shScram) and PC-depleted (shPC28) 4 T1 monolayers were wounded and closure was measured 15 and 24 h later. Values are presented as mean percent would closure, ±SEM. (PDF 372 kb

Additional file 2: of Pyruvate carboxylase supports the pulmonary tropism of metastatic breast cancer

Figure S2. PC expression in primary breast tumors. PC expression within tumor cells was rated as high, medium, low, or negative (n = 10). Representative sections from each group are shown. Data was obtained from the protein atlas dataset (www. proteinatlas.org ) [33]. (PDF 2805 kb