Journal: The Journal of Biological Chemistry

Article Title: Multi-modal mechanisms of the metastasis suppressor, NDRG1: Inhibition of WNT/β-catenin signaling by stabilization of protein kinase Cα

doi: 10.1016/j.jbc.2024.107417

Figure Lengend Snippet: Triple co-localization of β-catenin, NDRG1, and PKCα demonstrates the formation of a possible metabolon, decreasing β-catenin levels and nuclear localization. A – H , NDRG1 overexpression in PANC-1 cells significantly increases the co-localization between β-catenin, NDRG1, and PKCα. A and B , VC and NDRG1 overexpressing PANC-1 cells were incubated for 24 h/37 °C in the presence and absence of WNT3a (100 ng/ml). The cells were then examined for β-catenin ( green ), NDRG1 ( red ), and PKCα ( blue ) expression and triple co-localization ( white ) using confocal immunofluorescence microscopy. Studies were performed using a 100× objective at the same acquisition setting with Olympus Fluoview FV3000 software. Images were digitally magnified for better demonstration of the possible co-localization. The scale bar = 3 μm for all images except for the inset images, where the scale bar = 9 μm. C and D , deconvolution analysis was then implemented on the images in ( A and B ) using Olympus CellSens imaging software. White arrows demonstrate triple co-localization and possible formation of the metabolon. The scale bar = 3 μm for all deconvolution images except for the inset images, where the scale bar = 1.8 μm. In all studies, the images are representative of three experiments, and the quantitative analysis of the pixel intensities is shown for ( E ) β-catenin; ( F ) NDRG1; ( G ) PKCα; and ( H ) Co-localization between β-catenin, NDRG1, and PKCα. Quantitative analyses were performed using ImageJ software and were presented as the mean ± SD ( n = 3). Analysis of pixel intensity and co-localization were performed using 24 cells. Statistical significance is denoted as ∗∗ p < 0.01 and ∗∗∗ p < 0.001 comparing NDRG1 overexpressing PANC-1 cells in the presence and absence of WNT3a to VC cells in the absence of WNT3a; or ### p < 0.001 comparing NDRG1 overexpressing PANC-1 cells to VC cells in the presence of WNT3a.

Article Snippet: The images of visualized cells were then examined using Olympus Fluoview software, and in some studies, images were processed for deconvolution analysis with CellSens software (Olympus) to improve contrast and image resolution.

Techniques: Over Expression, Incubation, Expressing, Immunofluorescence, Microscopy, Software, Imaging