Fig 2: TACSTD2 increase and ICAM1 decrease of endogenous expression levels in DAPK1 ko clones.a qRT-PCR analysis of TACSTD2 (*P = 0.0204) and ICAM1 (***P < 0.001) gene expression in DAPK1 ko clones compared to wildtype HCT116 cells. B2M was used as reference gene. Three independent experiments per cell line are shown with one dot representing the mean of three technical replicates. Error bars represent mean ± SD. Unpaired t-tests were performed to compare qRT-PCR data. b Western Blot analysis of endogenous TACSTD2 and ICAM1 protein levels in HCT116 cells and DAPK1 ko clones. GAPDH served as loading control. Representative blots of two independent experiments are shown. c qRT-PCR of TACSTD2 (**P = 0.0082) and ICAM1 (***P < 0.001) gene expression in 5-day-old CAM micro-tumors. B2M was used as reference gene. Three independent experiments per cell line are shown with one dot representing the mean of three technical replicates. Error bars represent mean ± SD. Unpaired t-tests were performed to compare qRT-PCR data. d Western Blot analysis of TACSTD2 and ICAM1 expression in CAM tumor tissue (HCT116: 3 eggs, clone 7/6: 3 eggs, clone 10/8: 3 eggs). GAPDH served as loading control. e Representative images of ICAM1 and TACSTD2 immunohistochemistry of CAM micro-tumors (formalin-fixed and embedded in paraffin). Representative images are shown. Scale bar = 50 µm. ICAM1 immunoscores were obtained by multiplying staining intensity with percentage positivity divided by 10 (HCT116: n = 8; clone 7/6: 7, clone 10/8: 6, clone 21/9: 4). TACSTD2 immunoscores were obtained by multiplying staining intensity with percentage positivity (HCT116: n = 14; clone 7/6: n = 9; clone 10/8: n = 7; clone 21/9: n = 9). A Mann–Whitney test was performed to calculate significance between two groups (**P = 0.0037, ***P < 0.0001).

Fig 3: Visualization of suggested DAPK1/MAPK3(ERK2)/ICAM1/TACSTD2 signaling found via STRING database search of top 34 dysregulated genes of DAPK1 ko panel.Black star: additional input to STRING analysis. Color scheme: green: Notch1 cluster, blue: cluster of ECM modulators, red: cluster of more distant interactions. For full gene names and gene reference please refer to Table 1.

Fig 4: Downregulation of TROP2 promotes cell apoptosis(a) Flow cytometry was used to detect the rate of apoptosis in HepG2 cells following transfection with TROP2-siRNA sequence (40 pmol/mL) for 24 hours. (b) Flow cytometry was used to detect the rate of apoptosis in HCCLM3 cells following transfection with TROP2-siRNA sequence (40 pmol/mL) for 24 hours. The experiment was repeated three times; results are shown as mean ± standard deviation. **p < 0.01 vs. BC group.

Fig 5: Expression of Trop2 is reduced following transfection with TROP2-siRNA sequence(a) qPCR was performed to detect the mRNA level of Trop2 following transfection with TROP2-siRNA sequence (40 pmol/mL) for 24 hours in HepG2 cells. (b) Western blotting was performed to detect the protein level of Trop2 following transfection with TROP2-siRNA sequence (40 pmol/mL) for 24 hours in HepG2 cells. (c) qPCR was performed to detect the mRNA level of Trop2 following transfection with TROP2-siRNA sequence (40 pmol/mL) for 24 hours in HCCLM3 cells. (d) Western blotting was performed to detect the protein level of Trop2 following transfection with TROP2-siRNA sequence (40 pmol/mL) for 24 hours in HCCLM3 cells. The results are shown as mean ± standard deviation. *p < 0.05 and **p < 0.01 vs. BC group.