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.

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 [32] 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.

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 in vivo 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.

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 in vivo three dimensional time-lapse imaging of micrometastases in the chorioallantoic membrane of the avian embryo.

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.

Fluorescence in vitro 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 (Figure 5D, right) represents data from three independent western immunoblot experiments.

231LN cells expressing tdTomato (red) and tunable E-cadherin-zsGreen-DD (green) were injected intravenously in the avian embryo and allowed to extravasate and proliferate into micrometastases. Representative maximum intensity projections are shown. A) In vivo treatment with 1.0 µM Shield-1 demonstrates transition from a mesenchymal morphology to an epithelial morphology and continued maintenance of the epithelial morphology over an extended period of time (>40 hrs). Formation of E-cadherin junctions is apparent at t = 0.5 hrs, increasing through 24 hrs. B) Representative micrometastatic colony expressing tunable E-cadherin-zsG-DD and treated with 0.2 µM Shield-1. No induction effect is observed with 0.2 µM Shield-1 in vivo. C) Single Z-plane slices of a representative micrometastastic colony expressing tunable E-cadherin-zsG-DD and treated with 0.5 µM Shield-1. These panels represent the stabilization effect induced by 0.5 µM Shield-1 over the 12 hour time course; E-cadh-zsG-DD is stabilized and junctions appear between 231LN cells. E-cadh-zsG-DD junctions between cells of the micrometastatic colony are highlighted by arrows. D) Panels represent the depletion effect in the same colony with depleted levels of 0.5 µM Shield-1; E-cadh-zsG-DD junctions gradually disperse over time and 231LN cells eventually revert to a mesenchymal morphology. All scale bars are 25 µm. E) Quantitation of E-cadh-zsG-DD signal in 231LN-tdTomato cells in a 4-dimension image set over the entire 28 hour time course.