Fig 2: Ang1 induces ARP2/3 expression in colorectal cancer cells in vitro. (a–c) Western blotting of ARP2/3 expression in colorectal cancer (COLO320dm, SW620 and MC38) cells in the presence or absence of Ang1 (top panel). The intensity of ARP2/3 bands (n = 3) were quantified and normalized against GAPDH using ImageJ and represented as a fold change (bottom panel).

Fig 3: TGFß1 promotes cancer cells motility through RUNX1.a Western blot of ARP2/3 and vimentin expression in HT29 or LS174 cells upon exposure to recombinant TGFß1 (left panel). The right panel represents the intensity of ARP2/3 and vimentin bands (n = 3). b Immunofluorescence staining of colorectal HT29 cancer cells showing the effect of RUNX1 inhibitor (Ro5-3335, 0.5 µM) on the expression of ARP2/3 (green) and vimentin (red) in the presence of TGFß1 (100pM) or co-cultured hepatocyte (IHH) cell line. c Immunofluorescence staining of ARP2/3 (green) and vimentin (red) in colorectal cancer (HT29) cells expressing either scrambled or RUNX1 shRNA. The cells were either treated with TGFß1 (100pM) or co-cultured with a hepatocyte (IHH) cell line for 24 h. d Representative scratch assay in colorectal cancer (HT29, SW620, and COLO320dm) cells upon treatment with TGFß1 (100pM) individually or combined TGFß1 (100pM) plus RUNX1 inhibitor (Ro5-3335, 0.5 µM). e Immunoblotting showing ARP2/3 expression in RUNX1-silenced HT29 cancer cells expressing ectopic RUNX1 (top panel). The intensity of the bands was quantified using ImageJ and represented as a fold change. n = 3 independent experiments (bottom panel). f Scratch assay showing the rescue effects of RUNX1 in HT29 cancer cells invasion. Data are presented as the mean ± SD.

Fig 4: The expression of RUNX1 in cancer cells is crucial for interactions between cancer cells and hepatocytes. (a) represents immunoblotting of RUNX1 in HT29 cancer cells expressing either shRNA-scrambled or shRNA-RUNX1. (b) Cell viability was assessed with MTT assay in IHH cells cocultured with HT29 colorectal cancer cells expressing shRNA-scrambled or shRNA-RUNX1. (c–e) represent co-IF of IHH hepatocytes cocultured with HT29 cancer cells expressing shRNA control or shRNA against RUNX1. The cells were stained with HSA and cleaved caspase-3, HSA and cleaved PARP-1, or ARP2/3 and vimentin, respectively. Average pixel intensity was measured from three randomly selected areas for each sample. The right panels represent the quantification of positive pixels. Results are representative of 3 independent experiments. The bar chart shows mean values ± SD. ns = Not significant.

Fig 5: ARP2/3 mediates Ang1-driven cancer cell motility. (a,b) Western blotting of ARP2/3 expression in SW620 or COLO320dm cancer cells expressing shRNA-scrambled or shRNA-ARPC3. The right panels show the intensity of ARP2/3 bands that were quantified and normalized against GAPDH using ImageJ and represented as a fold change (n = 3). (c,d) Representative scratch assay in SW620 or COLO320dm cells expressing shRNA-scrambled or shRNA-ARPC3 upon exposure to Ang1. The right panels show the corresponding wound healing ratio shown in fold change (n = 3). Data are presented as the mean ± SD. ns = Not significant.