Fig. 4

MIEN1 knockout modulates key actin cytoskeleton proteins. (A) Immunofluorescence staining images of the actin cytoskeleton and pFAK. (B) Quantification of the intensity of pFAK-Y397 at the cell periphery and in the cell body of the same images, performed using ImageJ software. The paired t-test was used to compare the groups, with n = 2 in each group. (C) Immunofluorescence staining images of the actin cytoskeleton and pCofilin-S3. (D) Quantification of the intensity of pCofilin-S3 in HT29-WT and KO cells, performed using ImageJ software. The two-tailed unpaired t-test was used to compare the groups, with n = 6 in each group. (E) Western blot image representative of the whole-cell lysate of HT29 WT and KO cell lines, showing the expression of key cytoskeletal proteins, with Hsc70 as a control. (F) Quantification of the band density of western blot images for proteins to compare HT29 WT and KO cell expression, as performed using the ImageJ software. The paired t-test was used to perform statistical analysis, with n = 3 in each group. GraphPad Prism 10 was used to generate the graph and perform statistical analysis, with *, p < 0.05; **, p < 0.005; ***, p < 0.0005. Images were acquired using the Zeiss LSM 880 Confocal with Airyscan microscope at 63 × magnification. Fluorescence intensities were measured using ImageJ software. MIEN1, migration and invasion enhancer 1; KO, knockout; WT, wild-type.