TGFb1 each time. The harvested treated and non-treated cells after trypsinization were stained for flow cytometry at a concentration of 16106 cells per 100 ml of buffer (PBS pH 7.4, 2% PBS, 2 mM EDTA). Cells were incubated with conjugated antibodies (2 mg/106 cells) against MDR (Anti MDR-alexafluor 405, Santa Cruz Biotechnology) and Sca-1 (anti Sca-1-PE, BD Biosciences) at 4uC for 20 min. Fluorescence activated cell sorting (FACS) analysis was carried out using FACSAria flow cytometer (Becton Dickinson) at the core imaging facility of the UT Health Science Center at San Antonio, Texas. Cell proliferation assay 4T1 cells pre-treated with TGFb1 for seven days were plated in a 96-well plate at 2,000 cells/well. Cells were treated with doxorubicin at two different concentrations (1 nM and 10 nM) in the presence or absence of TGFb1 (5 ng/ml) and 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was utilized to obtain the relative cell number after 5 days of incubation as described previously [22]. Immunofluorescence The treated and non-treated cells (as described earlier) were harvested and about 46103 cells were seeded on a 8-well Lab-Tek chambered coverglass (Nunc International, Rochester, NY) and stained with Rhodamine phalloidin (Molecular probes) for F-actin or antibodies to Snail (Cell Signaling) after 24 hours. Briefly, for F- actin staining, cells were fixed in 4% paraformaldehyde, permeabilized in 0.1% Triton-X100 and incubated with Rhoda- mine phalloidine (1:100 dilution) for 30 min. For the staining with Snail antibody, cells were fixed in 4% paraformaldehyde for 15 min, incubated with 5% normal goat serum in PBS/Triton- X100 for 60 min, with primary antibody at 4uC overnight, and in Alexafluor 568 fluochrome-conjugated secondary antibody for 1 hr at room temperature. The slides were mounted with vectashield mounting media (Vector) and viewed under an Olympus Fluoview FV1000 confocal fluorescence microscope. Animal Studies Four-week-old female BALB/c and athymic nude mice (obtained from Harlan Sprague Dawley, Inc. Indianapolis, IN) were used for in vivo animal experiments. The animals were housed under specific pathogen free conditions. We investigated the effects of doxorubicin and/or TbRI-KI treatment on orthotopic xenograft tumors formed by murine 4T1 or human MDA-MB- 231 breast cancer cell lines, and also on their subsequent spontaneous lung and/or bone metastasis. Murine breast cancer 4T1 cells (0.16106) and human breast cancer EGFP expressing MDA-MB-231 cells (1.06106) were inoculated at the both inguinal mammary fat pad of syngeneic BALB/c mice and athymic nude mice respectively. In the study with 4T1 xenograft model, animals were treated with Doxorubicin (once a week via intraperitoneal (i.p.) or intravenous (i.v.) injection in two doses of 4 mg/kg and 8 mg/kg animal body weight respectively) and TbRI-KI (every alternative day via i.p. injection of 1 mg/kg body wt) either alone or in combination in two sets of experiment. In the first experiment, treatment started immediately after tumor cell inoculation and in the second experiment, treatment started when the average tumor volume per mouse was between 60–70 mm3. In the study with MDA-MB-231 cells, treatment of doxorubicin and TbR1-KI with similar doses as described above was started when the average tumor volume was between 80–100 mm3/mouse. Placebo control group received PBS every alternate day. In both studies, animals were ranked in closely matched groups according to their tumor volumes. Tumor sizes were measured twice weekly Doxorubicin and TGFb Inhibitor with a caliper, and the tumor volumes were calculated with the equation V = (L6W2)60.5, where L is length and W is width of a tumor. In all studies, animals were sacrificed after 21 days of treatment. In vivo spontaneous lung and bone metastasis assay After the termination of the all in vivo experiments, lungs were excised and metastatic cancer cell colonies were visually observed. In the 4T1 study, lungs were fixed in Bouin’s solution (Sigma) and surface lung nodules were counted. In the MDA-MB-231 study, EGFP expressing green lung metastatic colonies were observed and counted under a fluorescence microscope followed by 10% buffered formalin fixation. All lung specimens were embedded in paraffin and stained with hematoxylene and eosin. To analyze spontaneous bone metastasis in the 4T1 model, bone tissues (tibia and femora) were fixed in 10% neutral buffered formalin (Fisher Scientific) for 48 hours at room temperature, decalcified in 10% EDTA and embedded in paraffin. Sections were stained with hematoxylin, eosin, orange G and phloxine. The tumor burden in femora and tibia was examined microscopically. Statistical analysis Student t tests were used for the comparison between two mean values. For the comparison of more than two mean values we used one-way analysis of variance (ANOVA) followed with Newman- Keuls Multiple Comparison Test. All statistical analysis was performed using Prism version 3.03 software (GraphPad Software Inc.). Supporting Information Figure S1 TGFb and doxorubicin stimulated invasion of murine and human breast cancer cells. In a Boyden chamber invasion assay, 50,000 murine breast cancer 4T1 cells (A) and human breast cancer MDA-MB-231 cells (B) in serum free medium were placed inside the insert of an invasion chamber coated with matrigel and treated with doxorubicin (100 nM) or TGFb1 (5 ng/ ml in A and 1 ng/ml in B) for 18 hours and 24 hours respectively. Lower chamber contained the complete medium with 10% serum as chemoattractant. Stained membranes were counted under a microscope. Results are the mean+SEM of invasive cell numbers in three fields of observation at 1006 magnification in A and total number of invasive cells in B. Found at: doi:10.1371/journal.pone.0010365.s001 (0.09 MB TIF) Figure S2 Doxorubicin treatment enhanced TGFb signaling in murine and human breast cancer cells. A. The murine 4T1 and human MDA-MB-231 breast cancer cells were treated with doxorubicin (25 nM) or TGFb1 (2 ng/ml) in the presence or absence of TGFb antagonist TbRI-KI (500 nM) for 24 hours. The cell extracts were used for Western blotting analysis to measure the levels of phosphorylated Smad3 (P-Smad3). B. Mink lung epithelial cells stably transfected with TGFb-responsive PAI-1 promoter-luciferase reporter construct were plated in a 96-well plate and treated with doxorubicin or TGFb1 (0.5 ng/ml) for 24 hr. Luciferase activity was measured in cell lysate. Data are mean6SEM of 3 wells. Found at: doi:10.1371/journal.pone.0010365.s002 (0.09 MB TIF) Figure S3 Doxorubicin induced EMT in human breast cancer MDA-MB-231 cells. MDA-MB-231 cells were treated with TGFb1 (2 ng/ml) or doxorubicin (25 nM) in the presence or absence of TbRI-KI (500 nM) for 7 days in an 8-well Lab-Tek chambered coverglass (Nunc International, Rochester, NY). Cells PLoS ONE | www.plosone.org 11 April 2010 | Volume 5 | Issue 4 | e10365