DOI: 10.6084/m9.figshare.14532639.v1
title: Additional file 1 of Pals1 prevents Rac1-dependent colorectal cancer cell metastasis by inhibiting Arf6

description: Additional file 1: Supplementary Fig. 1. Pals1-deficient HCT116 cells do not exhibit defects in cell-cell contacts but increased motility. a Western blot analysis of the expression level of Pals1 in HCT116 and CRISPR/Cas9 generated HCT116ΔPals1 cell line. b-d Immunostainings of confluent HCT116 and HCT116ΔPals1 cells stained against Pals1 (green in b and c), PATJ (red in b), Claudin7 (magenta in c), ZO-1 (red in c) and E-Cad (green in d). The ratio of membranous versus cytosolic E-Cad was quantified (N = 50). e Western blot analysis of the protein expression of Pals1 and E-Cadherin in HCT116 and HCT116ΔPals1 cells. f Live-cell imaging of individual cell migration trajectories of HCT116 and HCT116ΔPals1 on basal membrane matrix coated surface over 5 h. g Quantification of the velocity of the single cell tracking experiments (N = 60). h Quantification of the translocation of the single cell tracking experiments (N = 60). i Proliferation of HCT116 and HCT116ΔPals1 was evaluated over 8 days using an automated cell counter (N = 3). j Staining of confluent HCT116wt and HCT116ΔPals1 for DAPI (blue) and TUNEL (red) in order to detect apoptosis. Quantification of TUNEL-positive cells gave a mean of 0.23 ± 0.13% for wt and 0.52 ± 0.18% for Pals1-deficient cells (N = 3). Scale bars are 20 μm. Supplementary Fig. 2. Knockout of Pals1 results in increased Arf6 but not Arf1 expression. a Quantification of active Cdc42 from pulldown assays (N = 3). b Western blot analysis of phosphorylated PAK1/2, which is induced by active Rac1. c Real time quantitative PCR analysis of the mRNA expression of Arf6 in HCT116 and HCT116ΔPals1 cells (N = 3). d Quantification of active Arf6 normalized against total Arf6 from pulldown assays (N = 3). e Immunostaining of migrating HCT116wt cells with anti Pals1 (green), anti Arf6 (red) antibodies and Phalloidin-staining (magenta) in order to visualize F-actin. Arrow indicates lamellipodium, arrowhead points at a cell-cell-contact. f Western blot and quantification of pulldown assays for active Arf1 using GST-GGA3 (N = 3). GST-GGA3 is visualized by CBB staining. g Western blot demonstrating efficient knockdown of Arf6 in Pals1-deficient HCT116 cells by two different shRNAs against Arf6. A scrambled shRNA (scr) was used as control. h Wound healing assay with HCT116∆Pals1 cells expressing scrambled shRNA or Arf6 shRNAs. (N &gt; 8). Scale bar is 20 μm. Supplementary Fig. 3. Knockout of Pals1 in SW48 cells results in increased cell migration and enhanced Arf6/Rac1 activation. a Western blot demonstrating the efficient knockout of Pals1 in SW48 cells. b Quantification of wound healing assays of SW48 and SW48ΔPals1 cell lines (N = 3). c and d Western blot analysis and quantification of GST pulldown assays for active Rac1 (N = 3) (c) and active Arf6 (N = 4) (d). e Quantification of Arf6 expression in SW48 and SW48∆Pals1 cells normalized against β-Actin. f Expression of Arf6 mRNA in HCT116 and HCT116∆Pals1 cells incubated with either DMSO, the Arf6 inhibitor NAV2729 (10 μM) or the ERK-inhibitor ERK U0126 (10 μM) for 24 h (N = 3). g Western blot analysis of wild type and HCT116∆Pals1 cells transiently transfected with empty vector or indicated Pals1 constructs. h Quantification of wound healing assays of wild type HCT116 cell and HCT116ΔPals1 expressing either GFP alone (control) or indicated Pals1-GFP variants (N = 5). Supplementary Movie 1 and 2. Wild type HCT116 cells (movie 1) and Pals1-deficient HCT116 cells (movie 2) were seeded (20,000 cells / ml) 2 days before the experiment on a matrix containing laminin, fibronectin and collagen IV. The motility was visualized by recording pictures every 5 min over 5 h.