Fig. 2: CEACAM6 knockdown inhibits GBC oncogenic phenotype.

A CEACAM6 knockdown using two siRNAs targeting CEACAM6 (si#2 and si#3) transiently transfected in SNU308 and Mz-ChA-1 are shown in Western blot. β-Actin served as loading control. B qPCR was performed to analyze CEACAM6 gene expression levels on SNU308 and Mz-ChA-1 cells. SRSF4 gene expression was used as internal control for normalization. C Three days after transfection, cells were used for adhesion assay and images of adherent SNU308 cells 1 h after seeding were captured and quantified. D Transwell migration assay of SNU308 cells was quantified by the area of migrated cells per image and representative images of 10× microscope magnification are shown. Mean results of three independent replicates are shown. E Bar graphs showing relative cell proliferation based on BrdU incorporation ELISA and F cell viability of SNU308 cells after 3, 4, and 5 days of CEACAM6 knockdown. G Cell cycle distribution in sub-G0, G0/G1, S, and G2/M phases of SNU308 cells after CEACAM6 knockdown is depicted. H Relative light unit (RLU) of Annexin V apoptosis assay after CEACAM6 knockdown in SNU308 cells. I Percentage of γ-H2AX positive cells and representative immunofluorescence images of SNU308 after CEACAM6 knockdown. The scale is 10 µm. Staurosporine (25 µM) treatment for two hours was used as positive control. J Western blot image of CEACAM6 and PARP protein after CEACAM6 knockdown or after two hours of Staurosporine (25 µM) treatment. β-Actin served as loading control. K The senescent cell area in SNU308 cells was captured based on β-galactosidase staining with ×10 magnification. Representative images are shown of three independent replicates. Inset pictures show a detailed area and the scale bar is 20 µm. L Quantification and representative images of colony area after 14 days of CEACAM6 knockdown in SNU308 cells. Data are represented as mean ± SD of three independent experiments. P values were determined by unpaired t-test (p ≥ 0.05 ns, <0.05 *, <0.01 **, <0.001 ***).