Journal: Cancers

Article Title: SCAMP3 Regulates EGFR and Promotes Proliferation and Migration of Triple-Negative Breast Cancer Cells through the Modulation of AKT, ERK, and STAT3 Signaling Pathways

doi: 10.3390/cancers14112807

Figure Lengend Snippet: SCAMP3 colocalizes with EGFR after receptor internalization. Cells were stimulated with 10 ng/mL EGF at the indicated time points to evaluate the location of SCAMP3 and EGFR using confocal microscopy. Representative images of the internalization assay and fluorescence quantification of SCAMP3, EGFR, and their colocalization in ( A – C ) SUM-149 and ( D – F ) MDA-MB-468 cells. The nuclei were stained with DAPI (blue). Secondary antibodies conjugated to Alexa 488 (green) and Alexa 594 (red) were used to detect SCAMP3 and EGFR, respectively. The micrographs were taken at a magnification of 60× using confocal microscopy. The white arrows indicate the colocalization of EGFR and SCAMP3 in the zoom images. The zoom-in of each image is shown in white squares and each has equal dimensions. Scale bar = 20 µm. Total cell fluorescence and colocalization area analyses were performed in 20 cells from three experiments using Image J. Colocalization was calculated as the ratio of the colocalization fluorescence area to the total cell fluorescence area. One way or two-way ANOVA; * p ≤ 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. Data are represented as mean ± SEM.

Article Snippet: After washing, coverslips were incubated with anti-rabbit Alexa 488 (1:750, #4412, Cell Signaling Technology (CST), Danvers, MA, USA) and anti-mouse Alexa 594 (1:750, #8527, CST) for 1 h at room temperature.

Techniques: Confocal Microscopy, Fluorescence, Staining