Fig 2: Competitive inhibition of TWIST1 WR domain binding. a Schematic representation of GFP alleles used. GFP contains 23 amino acids encoded by the multiple cloning site of the vector at its C-terminus. GFP-WR contains the first two such amino acids (Leu-Glu encoded by XhoI restriction site), followed by the 20 amino acids of the WR domain. Thus, the two alleles have indistinguishable molecular weights. b Left, fluorescent microscopy shows that GFP and GFP-WR are expressed at similar levels and in similar patterns in HEK-293 cells. Scale bar, 100 µm. Right, Western blot confirms equal GFP and GFP-WR expression. c CoIP with RELA pulldown reveals that in the presence of increasing GFP-WR expression, TWIST1-RELA binding is reduced in a dose-dependent manner. d CoIP with GFP pulldown reveals that increasing GFP-WR dose results in more TWIST1 co-precipitated with GFP. Graph represents ratio of TWIST1 to GFP, normalized to their respective inputs. Error bars, standard deviation of duplicate experiments. e Dual luciferase assay demonstrates that as seen in the RELA CoIP, there is a dose dependent drop in IL-8 driven luciferase expression with increasing dose of GFP-WR inhibitor. Graph represents firefly luciferase expression normalized to renilla luciferase for each condition. Error bars represent standard deviation of biological triplicate experiments. GFP without GFP-WR condition was used as the basis for statistical comparisons. pGL3 lacking the IL-8 promoter was used as a negative control. ****, p < .0001

Fig 3: Proliferation occurs throughout Twist1+ cell dissemination. (A-D) To monitor for proliferation events during dissemination, Adeno-CMV-Cre was used to induce low-level genetic mosaic activation of Twist1 in isolated R26::LSL-rtTA;TRE-Twist1;mT/mG organoids. Individual Twist1+ cells were tracked in real-time by confocal microscopy. Cell division was observed during basal migration (A, n=12 divisions in 12 organoids from three biological replicates); during cell release from the epithelium (B, n=6 divisions in five organoids from three biological replicates); and during migration in the ECM (C, n=9 divisions in six organoids from two biological replicates). White and blue asterisks in A-C denote daughter cells. Panels in A' depict 3D reconstructions of the cell surfaces during cell division. The final positions of the daughter cells in A' are displaced in the z-axis. White arrowhead in A' indicates the point of cell division. Scale bars: 10 µm. (E,F) To test for the requirement for proliferation, CMV::rtTA;TRE-Twist1 organoids were cultured with aphidicolin. Panels in F show representative images of organoids cultured with vehicle (DMSO), 100 nM aphidicolin, and 10 µM aphidicolin. Red arrowheads in E indicate disseminated cells. (F) The dot plot shows dissemination normalized to the median number of disseminated cells in the vehicle control. Each dot represents an organoid, pooled across three independent biological replicates. Dissemination was not significantly reduced at either 100 nM (P>0.9999; n=84 organoids) or 10 µM (P>0.9999; n=54 organoids) aphidicolin as compared to control (n=89 organoids). Scale bars: 50 µm. Veh, vehicle; ns, not significant.

Fig 4: Metastasis behavior of cisplatin-resistant and parental OC cells. (A) Twist expression in CisR and parental OC cells. (B) Migration ability of cisplatin-resistant and parental OC cells determined by transwell migration assay (Magnification, 10×, scale bar 100 µm). (C) Wound healing capability of CisR and parental OC cells (Magnification, 10×, scale bar 100 µm). Values were represented as mean ± SD (n = 3). # p < 0.05, compared with the parental group.

Fig 5: CADM1 is negatively regulated by TWIST1 in human melanoma cell lines.a Validation of microarray via qRT-PCR analysis. b-c qRT-PCR (b) and protein levels (c) of TWIST1 and CADM1 after control or TWIST1 siRNA transfections in WM793 cells. Densitometry for the ratio of CADM1 to ERK2 is indicated. d Lysates were probed via Western blotting after knockdown of TWIST1 levels by siRNA transfections in the mutant BRAF melanoma cell lines M229 and M238, and the mutant NRAS cell lines, Sbcl2 and WM1366. TWIST1 and CADM1 protein levels were assayed with ERK2 as a loading control. e qRT-PCR levels of CADM1 in A375TR cells after induction of either control LacZ or TWIST1. f Reporter constructs consisting of either an empty vector control or 1047bp upstream of CADM1 putative start site were transfected into WM793TR LacZ or WM793TR TWIST1 cells. Luciferase output was measured as an indication of promoter activity. Results are averages from three independent repeated experiments. Fold luciferase output is normalized to the levels of WM793TR LacZ cells per each luciferase construct. * represents p < 0.05 as determined by t-test, error bars are ±SEM. g CADM1 protein levels were measured via western blot after 96 h of exogenous TWIST1 expression. h Chromatin immunoprecipitation (ChIP) was performed on WM793 cells samples with TWIST1 antibody or V5 epitope antibody as a negative control. Associated genomic DNA was then subjected to qRT-PCR to determine the enrichment of each area of interest indicated in Supplementary Fig. 2. Results are averages from three independent repeated experiments. * represents p < 0.05 as determined by t-test, error bars are ±SEM