Loss of Neogenin1 in human colorectal carcinoma cells causes a partial EMT and wound-healing response

Neogenin1 (NEO1) is a receptor of the Deleted in Colorectal Carcinoma (DCC)/Frazzled/UNC-40 family, which regulates axon guidance but can also stabilize epithelial adherens junctions. NEO1 and DCC are also tumor suppressors that can inhibit metastasis by acting as dependence receptors. Given the role of NEO1 in maintaining adherens junctions we tested whether loss of NEO1 also promoted metastasis via an epithelial mesenchymal transition (EMT). Loss of NEO1 disrupted zonula adherens but tight junctions were unaffected. Neo1-depleted epithelial cells exhibited a more migratory morphology, had reduced F-actin rich stress-fibres and more basal lamellipodia. Microtubule density was decreased while microtubule outgrowth was faster. Live imaging showed that Neo1-depleted epithelial islands had increased lateral movement. Western blots and immunostaining revealed increased expression of mesenchymal markers such as Fibronectin and MMP1. Furthermore, RNA-seq analysis showed a striking decrease in expression of genes associated with oxidative phosphorylation, and increased expression of genes associated with EMT, locomotion, and wound-healing. In summary, loss of NEO1 in intestinal epithelial cells produces a partial EMT response, based on gene expression, cellular morphology and behaviour and cytoskeletal distribution. These results suggest that loss of NEO1 in carcinomas may contribute to metastasis by promoting a partial EMT and increased motility.

Supplementary Figure S1 Neo1 knockdown in Caco-2 cells was confirmed by Western blot analysis. Representative blot with three biological replicates from one experiment. The boxed region has been shown in Figure 1. Same blot has been used for GAPDH probing after being stripped using a mild stripping buffer (Abcam) Supplementary Figure S2 No significant change in E-Cad protein levels after Neo1 knockdown. Each band represents cell lysate proteins from a biological replicate from three independent experiments. The boxed region has been shown in Figure 1 and the blot has been scanned at high and low contrast. Same blot has been used for GAPDH probing after being stripped using a mild stripping buffer (Abcam)

Supplementary Figure S3
Western blot for ZO-1 in control and Neo1-siRNA treated cells. The boxed region has been shown in Figure 1 and the blot has been scanned at high and low contrast. Blot outline has been marked using dotted lines Supplementary Figure S4 a. DLD-1, RKO, and SW480 cells treated with control or Neo1-siRNA and stained for F-Actin and immunostained for E-Cad. siRNA was transfected into 1day old epithelia and cells stained after 2 days. Scale bar-20µm. b. RT-qPCR of Neo relative to the reference gene (TBP) in different CRC lines. Error bars show SEM; p-values based on two-tailed student's t-test relative to Caco-2 levels.
Supplementary Figure S5 Significant upregulation of fibronectin protein levels after Neo1 knockdown. Each band represents cell lysate proteins from a biological replicate from three independent experiments. The boxed region has been shown in Figure 4 and the blot has been scanned at high and low contrast. Same blot has been used for GAPDH probing after being stripped using a mild stripping buffer (Abcam). Each band represents cell lysate proteins from a biological replicate from one experiment. The boxed region has been shown in Figure 6 and the blot has been scanned at high and low contrast. Figure S8 RT-qPCR of control and Neo1 knockdown post-transfected Caco-2 cells shows significant downregulation of KRT8 and KRT18 in Neo1 knockdown cells suggesting partial EMT. Data represent mean ± SEM of 6 biological replicates, p=0.04 and p=0.01 respectively (two-tailed student's t-test)

Supplementary Figure S9
Kymograph analysis of control and Neo1-knockdown Caco-2 cells revealed an increase in the angle of movement of internal cellular features in Neo1 knockdown cells. Time-lapse movie images (i) captured at 15 sec intervals (ii) were resliced in the timedimension to create kymographs (iii), which were then analysed using ridge detection (iv) to detect lateral movement of internal features and the Feret angle for each ridge determined (v). See Materials and Methods for details.
Supplementary Movie S1 -Time-lapse imaging of control and Neo1 knockdown cells was performed and epithelial islands were imaged for a total of 30 min with an image every 15 sec. To quantify the lateral movement of cells, collected sequences were processed with an Image Stabilizer plugin (K. Li, http://www.cs.cmu.edu/~kangli/code/Image_Stabilizer.html), and then temporally smoothed by calculating a running average over 50 time-frames. The split panel shows an example of time lapse movie before and after smoothing. Supplementary Table S1 -Table of genes that were significantly upregulated and downregulated in Pathway enrichment analysis for Neo1 siRNA transfected cells at day 2 posttransfection. Pathway enrichment analysis was performed using iDEP v0.73. Table of genes that were significantly upregulated and downregulated in Pathway enrichment analysis in Neo1 siRNA transfected cells at day 5 after co-transfection. Pathway enrichment analysis was performed using iDEP v0.73. Table of genes           Kymograph construction