Figure 1.
WAVE2 knockdown uniquely results in aberrant 3D morphology of MCF10A epithelial cells.
A. Phase contrast images of control and WAVE2-KD cells grown in 2D and 3D cell culture (Day 16). B. Representative Western blots of WAVE2, Abi1 and Sra1 in control and WAVE2-KD cell lines. Numbers below the bands indicate average densitometry values from n = 3 blots normalized to loading control protein levels. Representative GAPDH loading control blot is shown. C. Quantification of average acini diameter. n = 30 acini of each cell type selected from 3 independent experiments, error bars represent standard error of the mean. * indicates p<0.05.
Figure 2.
Large multilobular WAVE2-KD acini arise from a single acinar structure.
Frames from the Supplemental Movies (S1–S3). Control and WAVE2-KD acini were imaged during 3D culture growth from days 4.5 to 7. Note the formation of lobules from round WAVE2-KD acini during this time period.
Figure 3.
Cellular proliferation is dysregulated in WAVE2-KD cells in 3D culture but not in 2D culture.
A. Control and WAVE2-KD acini were immunostained with Ki67 to identify proliferating cells (arrows) and counterstained with DAPI to show total number of nuclei. Single confocal slices through the central region of the acini are shown at Days 10 and 12 of growth. Scale bar 100 µm. B. Quantification of the number of Ki67 positive cells in acini at Days 10, 12 and 14 of growth. Error bars represent standard error of the mean. * indicates p<0.05. C. Quantification of average acini diameter over time. n = 10 acini of each cell type at each time point selected from 3 independent experiments, * indicates p<0.05. D. Control and WAVE2-KD acini were immunostained with activated Caspase-3 to identify apoptotic cells and counterstained with DAPI to show total number of nuclei. Single confocal slices through the central region of the acini are shown at Day 12 of growth. Scale bar 100 µm. E. Control and WAVE2-KD acini were immunostained with laminin-332 to indicate basal polarity. Scale bar 100 µm. F. Cell proliferation of control and WAVE2-KD cells in 2-dimensional cell culture. n = duplicate wells from 3 independent experiments (6). Error bars represent standard error of the mean. * indicates p<0.05.
Figure 4.
Loss of WAVE2 results in heterogeneous E-Cadherin localization at cell-cell adhesions.
A. Confocal images were captured through the center of control and WAVE2-KD acini immunostained for E-Cadherin (green) and counterstained with Alexa-568 phalloidin (red) to label actin filaments and DAPI (blue) to label nuclei. The white boxes in the low power merged images indicate location of the enlarged areas shown below in the lower panels. Note low and heterogeneous E-cadherin staining at actin-positive cell-cell junctions. Scale bar 50 µm. B. Quantification of the ratio of junctional E-Cadherin to actin. n = 50 cell-cell junctions. Error bars represent standard error of the mean, * indicates p<0.05.
Figure 5.
E-Cadherin is downregulated at the RNA level in WAVE2-KD cells and contributes to the aberrant WAVE2-KD acinus morphology.
A. Western blot of E-Cadherin levels within control and WAVE2-KD cells. GAPDH levels are shown as a loading control. Numbers below the bands indicate average densitometry values from n = 3 blots normalized to control protein levels. B. Quantitative real-time PCR data of E-Cadherin expression within control and WAVE2-KD cells. n = 3, *p<0.05, compared to control. C. Western blot analysis of N-Cadherin protein levels in control and WAVE2-KD cells. β-actin levels are shown as a loading control. Numbers below the bands indicate average densitometry values from n = 3 blots normalized to control protein levels. D. Quantitative real-time PCR data of N-Cadherin expression in control and WAVE2-KD cells. n = 3, *p<0.05, compared to control. E. Representative phase contrast images of control + E-Cadherin and WAVE2-KD + E-Cadherin cells in 2D and acini grown from those cells in 3D (Day 16). The fluorescence image inset shows GFP levels within the E-Cadherin rescued WAVE2-KD2 acini which is indicative of E-Cadherin levels within the acini. F. Quantification of the average acini diameter of control and WAVE2-KD acini expressing exogenous E-Cadherin. The WAVE2-KD acini were divided into 2 groups based on GFP expression and quantified separately. n = 20 acini of each cell type selected from 3 independent experiments, error bars represent standard error of the mean. * indicates p<0.05.
Figure 6.
WAVE2 functions upstream of Twist1 to control breast epithelial 3D morphology.
A. Quantitative real-time PCR data of Twist1 levels within control, WAVE2-KD cells +/− Twist1-KD. B. Quantitative real-time PCR data of E-Cadherin expression in control and WAVE2-KD cells as well as the respective Twist1-KD cells. C. Quantitative real-time PCR data of N-Cadherin expression in control and WAVE2-KD cells as well as the respective Twist1-KD cells. n = 3, * indicates p<0.05, compared to control. D. Phase contrast images of acini grown from control cells, control + Twist1-KD, and WAVE2-KD + Twist1-KD cells. E. Quantification of average acini diameter of Twist1 knockdown acini. n = 30 acini of each cell type selected from 3 independent experiments, error bars represent standard error of the mean. Values were not significantly different from control acini.
Figure 7.
Abl kinase-WAVE2 interactions are critical for regulation of epithelial morphology.
A. Western blot of phosphorylated CrkL (Y207) in control and WAVE2 knockdown cells and a western blot of GAPDH as a loading control. Numbers below the bands indicate average densitometry values of P-CrkL from n = 3 blots. B. Phase contrast images of acini from control MCF10A cells or MCF10A cells transfected with full length Abl kinase. C. Phase contrast images of control and WAVE2-KD acini that were grown in the presence of 10 µM STI571 or vehicle control. D. Quantification of average acini diameter of control and Abl overexpressing acini. n = 20 acini of each cell type selected from 3 independent experiments, error bars represent standard error of the mean. * indicates p<0.05. E. Quantification of average acini diameter of STI571 treated acini compared to untreated acini. n = 30 acini of each cell type selected from 3 independent experiments, error bars represent standard error of the mean. * indicates p<0.05.