Rapid induction of the fluorescent protein zsGreen in MDA-MB-231LN (231LN) cells <i>in vitro</i>.

<p>231LN cells containing both tdTomato and zsGreen-DD were grown on glass coverslips. Panels represent fluorescence time-lapse imaging of 231LN cells treated with vehicle (A) and 1.0 µM of Shield (B). C) Quantification of zsGreen signal within the cells in the presence and absence of Shield-1 over time (*denotes p<0.01 compared to Vehicle treatment kinetic, N>10 cells per field of view, 10 fields of view analyzed per group). Treatment with 0.5, 1.0 and 2.0 µM Shield-1 revealed similar first order kinetics, while treatment with 0.2 µM Shield-1 revealed a similarly steep but brief increase (induction) in signal accumulation followed by a less steep kinetic at 4 hours post-treatment (depletion kinetic). D) Fluorescence immunohistochemistry demonstrates co-localization of proteasome (α1-20S antibody in red) with zsGreen-DD signal in 231LN cells in the absence of Shield-1. All scale bars are 25 µm.</p

Time-lapse imaging of E-cadherin induction and kinetics of cell-cell junction formation <i>in vitro</i>.

<p>Fluorescence <i>in vitro</i> time-lapse imaging of 231LN cells containing inducible E-cadherin treated with vehicle (A) or 0.2 µM Shield-1 which will produce an induction and depletion effect over 24 hours (B). Data Scale bar is 25 µm. C) Measure of “actively engaged" E-cadherin in 231LN cells in the presence of varying levels of Shield-1 (0.2, 0.5, and 1.0 µM Shield-1), expressed as the cumulative length of all zsG-positive adherens junctions over time (µm/hrs) in representative time-lapse experiments. The black kinetic represents the total cumulative length of E-cadherin-based junctions within a field of view at that timepoint while the red kinetic represents the accumulation of zsGreen-DD exposed to similar Shield-1 treatment. The “induction” and “depletion” phases of chemical induction are annotated in each graph. D) Conditioned media collected from cells expressing pzsGreen-DD which were treated with 0.2 µM Shield for 0, 6 and 12 hours were used to induce E-cadherin-zsGreen-DD expression in 231LN cells expressing E-cad-zsG-DD. There is induction with the 0 and 6 hours conditioned media, but minimal effect with the 12 hour conditioned media. Conversely, conditioned media from cells treated with 5.0 µM Shield-1 induced E-cadherin-zsG-DD expression regardless of the time of conditioned media collection. Graph (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030177#pone-0030177-g005" target="_blank">Figure 5D</a>, right) represents data from three independent western immunoblot experiments.</p

Characterization of tunable E-cadherin-zsG-DD protein expression in 231LN cells <i>in vitro</i>.

<p>A) Representative images of 231LN cells expressing fluorescent E-cadherin chimeras. Cell nucleus as stained by Hoechst (blue), E-cadherin-zsGreen (green), and tdTomato to highlight the cytoplasm (red) reveal the changes in cell morphology when E-cadherin is over-expressed (row 2) or induced with Shield-1 for 12 hours (row 4) compared to control (row 1) or un-induced cells (row 3). Arrows (yellow) highlight junctions formed by Shield-1-stabilized E-cadh-zG-DD. Scale bars are 20 µm. Insets show magnified view (250%) of cellular junctions. B) Examples of circularity measurements of representative 231LN cells (left) and 231LN cells expressing E-cadh-zsG-DD treated with 1.0 µM Shield-1 (right). C) Circularity measurements to assess a mesenchymal vs. epithelial morphology in cells described above. N = 70 per group, * denotes p<0.01 between groups, 2-way ANOVA.</p

Intravital time-lapse imaging of fluorescent protein induction in 231LN cells <i>in vivo</i>.

<p>231LN cells expressing tdTomato (red) and inducible zsGreen-DD (green) were injected intravenously in the avian embryo and allowed to extravasate and proliferate into micrometastases. Representative time-lapse images (maximum intensity projections) are shown after intravenous administration of Vehicle (A), 0.2 µM Shield-1 (B), and 0.5 µM Shield-1 (C). D) Quantification of <i>in vivo</i> zsGreen fluorescence in tdTomato-positive cells over time. Data for Vehicle (black kinetic), 0.2 µM Shield-1 (red kinetic), 0.5 µM Shield-1 (green kinetic), and 1.0 µM Shield-1 (blue kinetic) are represented as averages of at least three movies analyzed in each group. Error bars are SE and scale bar represents 25 µm.</p

A chemically tunable form of E-cadherin for use in intravital imaging.

<p>A) Expression vectors encoding tunable zsGreen (pzsGreen-DD), fluorescent E-cadherin (pE-cadh-zsG) and tunable fluorescent E-cadherin (pE-cadh-zsG-DD). Components include CMV promoter (pCMV), zsGreen fluorescent protein (zsGreen), the Shield-1 binding degradation domain (FKBP-DD), and E-cadherin. B) Schematic of MDA-MB-231-luc-D3H2LN (231LN) cells used to express tunable proteins and the predicted behavior of cells in the presence or absence of Shield-1. 231LN tumor cells were stably transfected with tdTomato and zsGreen alone or as a fusion with E-cadherin. C) Intravital imaging platform (right) with avian embryo imaging chamber (left) to maintain proper temperature (37°C) and humidity (>90%) used to perform <i>in vivo</i> three dimensional time-lapse imaging of micrometastases in the chorioallantoic membrane of the avian embryo.</p

Induction of E-cadherin-zsG-DD protein in 231LN cells by Shield-1 ligand and expression of vimentin.

<p>A) 231LN cells expressing E-cadh-zsG-DD (green) treated with 1.0 µM Shield-1 for 24 hours and immunostained with anti-E-cadherin mAb (red) and Hoechst nuclear stain (blue). Scale bars are 25 µm. B) Western immunoblot analysis of E-cadherin expression in 231LN cells expressing E-cadh-zsG-DD and treated with 1.0 µM Shield-1 using the same mAb as in A). Graph (right) represents analyses performed on three independent induction experiments. Cell lysates of 231LN cells expressing E-cadherin-zsG are shown in the first lane. Lysates of cells expressing E-cadherin-zsG-DD were collected at 0, 4, 8, 12, 16, and 24 hrs after Shield-1 treatment (1.0 µM final), revealing accumulation of Shield-1 stabilized E-cadherin-zsG-DD within cells (∼135 kDa). Far right lane is a positive control of 21PT cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030177#pone.0030177-Souter1" target="_blank">[32]</a> which endogenously express high levels of E-cadherin (∼110 kDa). C) Western immunoblot analysis of markers for epithelial-mesenchymal transition (EMT). Blot (left panels) reveals a decrease in vimentin protein levels when E-cadh-zsG-DD is induced by 1.0 µM Shield-1 treatment. Graph (right) represents analyses performed on three independent induction experiments.</p

Radiation-induced lung injury after concurrent neoadjuvant chemoradiotherapy for locally advanced breast cancer

Radiation-induced lung injury after concurrent neoadjuvant chemoradiotherapy for locally advanced breast cance

Knock down of α9 integrin and VEGF-D in 468LN cells reduced primary tumor growth in nude mice:

<p>(<b>A</b>) Representative images of tumors and Matrigel alone as well as the draining lymph nodes on day 20 (a scale in mm shown in the background). (<b>B</b>) Tumor growth rates determined by volume measured externally were dramatically reduced in both α9 integrin (Δα9/468LN) and VEGF-D knock down (ΔVEGF-D/468LN) tumors (indistinguishable from Matrigel alone). (<b>C</b>) Tumors were excised and mean weights of tumors and Matrigel were retrieved on day 20. Weights of 468LN tumors were significantly higher than those of Matrigel alone or Δα9/468LN and ΔVEGF-D/468LN tumors. Data represented as means (n = 16 for tumors and 4 for Matrigel) ± S.E. *p<0.05, **p<0.001.</p

Metastatic spread of tumor cells to the draining lymph nodes is abrogated by α9 integrin and VEGF-D knock down in 468LN cells:

<p>(<b>A</b>) Metastasis to lymph nodes were validated by the visual localisation of many cancer cells in 468LN tumor draining nodes identified by the GFP-marker and their absence in lymph nodes draining α9 integrin and VEGF-D knock down tumor implants. Nuclei were stained blue by DAPI. (<b>B, C</b>) Above conclusions were further validated by measuring the human mitochondrial-specific marker MTCO2 at the mRNA level with qRT-PCR (<b>B</b>) and protein level with western blot (<b>C</b>) was high in lymph nodes draining 468LN tumors. This process was nearly completely abrogated in both Δα9/468LN and ΔVEGF-D/468LN tumor implants. Data were derived from 16 lymph nodes. Four lymph nodes draining into an individual tumor in a single mouse were pooled to extract RNA and proteins, showing the data in four mice; Matrigel was retrieved from four implants. Data represents mean (n = 4) ± S.E, *p<0.05, *p<0.001.</p

Reduction of migration and invasion of 468LN cells by knocking down VEGF-D production:

<p>(<b>A–C</b>) Levels of VEGF-D knock down in 468LN cells was confirmed by three different methods, <b>(A)</b> qRT-PCR, (<b>B</b>) western blot and (<b>C</b>) ELISA. After VEGF-D knock down (KD) in 468LN cells, (<b>D</b>) migration, invasion and (<b>E</b>) proliferation significantly dropped as compared to scrambled knock down (SCR-KD) cells. (<b>F</b>) Addition of exogenous rVEGF-D or FBS increased both migration and invasion of VEGF-D knocked down 468LN cells. Bars represent mean (n = 4 in all cases except <b>C</b>, n = 6) ± SE, *,P<0.05; **,P<0.01.</p