Fig 2: DLC1-START binds Phosphatidylserine and this binding improves its binding to Caveolin-1 or PLCD1. A Pull-down (PD) using control (ctrl) or PS-coated beads in H358 (top) or 293 T (bottom) cells overexpressing the indicated GST or GFP-fusion plasmids, followed by GST or GFP Western blot (WB). The correct expression of all plasmids was verified in total lysates by WB. B Immunoprecipitation (IP) in 293 T overexpressing GST Caveolin-1 (GST CAV) (top) or GFP DLC1 (bottom) using the indicated antibodies, in the presence or absence of 100 μM PS added to the binding reaction. Immunocomplexes were analyzed by Western blot (WB) using the indicated antibodies. C Immunoprecipitation (IP) in 293 T overexpressing GST PLCD1 (GST PLCD1) (top) or GFP DLC1 (bottom) using the indicated antibodies, in the presence or absence of 100 μM PS added to the binding reaction. Immunocomplexes were analyzed by Western blot (WB) using the indicated antibodies. D Pull-down (PD) using MBP control or MBP DLC1 START 848–1091 beads in 293 T overexpressing the indicated GST-fusion plasmids, in the presence or absence of 100 μM PS added to the binding reaction, followed by GST Western blot. The amount of MBP-purified proteins is shown as stained with Ponceau S (bottom panel). E Pull-down (PD) using control (ctrl) or PS-coated beads and MBP control or MBP DLC1 START 848–1091 purified proteins (eluted from amylose resin in the presence of maltose), followed by MBP Western blot (WB). The purity and amount of MBP-purified proteins is shown in the western blot from the inputs, which represents a fraction of the total purified protein used in the pull-down

Fig 3: Mapping of DLC1 and PLCD1 interacting regions. A Co-immunoprecipitation of endogenous PLCD1, DLC1 and Caveolin-1 proteins in H1703 cells. The indicated antibodies for immunoprecipitation (IP) and Western blot (WB) were used. Input represents a fraction of the H1703 cell lysate before immunoprecipitation. B Schematic representation of the domain organization of DLC1 (top) and DLC1 constructs cloned as GFP-fusion proteins for immunoprecipitation in C. C PLCD1 Immunoprecipitation (IP) in cell lysates from H358 cells overexpressing control (GFP) or GFP-fusion plasmids spanning several regions in DLC1 followed by Western blot (WB) using the indicated antibodies. The expression of all GFP-fusion plasmids in total lysates is shown in the bottom panel. Asterisks (*) indicate the expression of the corresponding GFP-fusion proteins. D MBP pull-down (PD) in 293 T cells expressing GST control or GST PLCD1 using MBP control or MBP-DLC1 START 848–1091 purified proteins (from E.Coli), followed by GST Western blot (WB). The amount of MBP-purified proteins is shown as stained with Ponceau S (bottom panel). Input represents a fraction of the 293 T cell lysates before pull-down. E Schematic representation of the domain organization of PLCD1 (top) and PLCD1 constructs cloned as GST-fusion proteins to be used in F and G. F Glutathione pull-down (PD) in 293 T cells transfected with GST-PLCD1 plasmids encompassing several PLCD1 domains and GFP or GFP DLC1, followed by GFP and GST western blot. The expression of all GFP-fusion plasmids in total lysates is shown (bottom). G Pull-down (PD) using MBP DLC1 START 848–1091(purified from E. coli) and GST or GST-PLCD1 domains, followed by GST western blot. The amount of MBP- purified protein is shown as stained with Ponceau S. Expression of GST fusion proteins was checked by GST western blot in total lysates (bottom)

Fig 4: Caveolin-1 and PLCD1 cooperate with DLC1 to inhibit cell migration but their binding to DLC1 is mutually exclusive. A Migration in transwells of stable H358 control (V) or DLC1-overexpressing cells transiently transfected with GST control, GST Caveolin-1 (GST CAV1), GST PLCD1 or GST CAV1 + GST PLCD1. Migrated cells were fixed and photographed (top panel shows a representative image from each condition). The number of migrated cells were quantified using Image J (middle panel), and the percentage (%) of inhibition was calculated from a total of four random images taken from each experiment duplicate. Bars represent mean +/− SD. t-test was performed for statistical analysis (***:p < 0.005). The correct expression of GST proteins and DLC1 was checked by Western blot (WB). B PLCD1 Immunoprecipitation in H1703 cells transfected with GST control or GST Caveolin-1 (GST CAV1) plasmids, followed by Western blot (WB) using the indicated antibodies. C Caveolin-1 (CAV1) Immunoprecipitation (IP) in H1703 cells transfected with GST control or GST PLCD1 plasmids, followed by Western blot (WB) using the indicated antibodies. The correct expression of GST fusion proteins in B and C is shown from total lysates (bottom panels). D Immunoprecipitation (IP) using PLCD1 (top) or Caveolin-1 (CAV1, middle) antibodies in H358 cells transfected with empty vector (V) or pCDNA DLC1, followed by Western blot (WB) using the indicated antibodies. Correct expression of proteins in total lysates is shown in the bottom panel. E PLCD1 Immunoprecipitation (IP) in H358 cells transfected with empty vector (V) or a plasmid encoding the DLC1 START domain-containing region (DLC1 848–1091), followed by Western blot (WB) using the indicated antibodies. The correct expression of all proteins in total lysates is shown (bottom panel). F Caveolin-1 Immunoprecipitation (IP) in H1703 cells transfected with GFP or GFP-DLC1 START domain containing region (848–1091), followed by Western blot (WB) using the indicated antibodies. The correct expression of proteins in total lysates is shown (bottom panel)

Fig 5: All colorectal-cancer TCGA DLC1 START mutations show impaired inhibition of migration and anchorage-independent growth, and deficient binding to some or all START binding partners, but intact RhoGAP activity. A Migration in transwells of H358 control (V) or DLC1 wild type (WT) and mutants stable clones. Migrated cells were fixed and photographed (upper panel), and the number of migrated cells were quantified using Image J, from a total of four images from each duplicate experiment (bottom panel). Bars represent mean +/−SD. t-test was performed for statistical analysis (***, ###:p < 0.001) B. Same stable clones as in A were seeded in soft agar and allow to form colonies for approximately 28 days, and those colonies > 0.2 mm were photographed (upper panel) and counted (lower panel). Each dot represents one plate of cells. 3–5 plates of each stable clone were used for the experiment. Bars represent mean +/− SD. t-test was performed for statistical analysis (***:p < 0.001, ###:p < 0.005) C. Active Rho (GTP-RhoA) in 293H control (V) or DLC1 wild type (WT) and mutants transient transfectants was analyzed by Rhotekin pull down assays, followed by RhoA Western blot (WB).GFP DLC1 and total Rho expression were also checked by WB. D Glutathione Pull-down (PD) in 293 T cells overexpressing GST control or GST Caveolin-1 (GST CAV) and GFP control (V) or GFP DLC1 wild type (WT) and mutants followed by Western blot (WB) using GFP and GST antibodies. Correct expression of GFP fusion proteins was checked in total lysates (bottom panel). E PLCD1 immunoprecipitation (IP) in 293 T cells overexpressing GFP control (V) or GFP DLC1 wild type (WT) or mutants followed by western blot (WB) using the indicated antibodies. The expression of GFP fusion proteins was checked by Western blot in total lysates (bottom panels)