Fig 2: Knockdown of SCAMP3 expression affected cell cycle of HCC cell lines(A-C) The proportions of Huh7 (A), PLC5 (B), Hep3B (C) cells in G1 phase increased significantly in SCAMP3-siRNA group in comparison with NC group.

Fig 3: 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.

Fig 4: The expression of SCAMP3 in HCC and adjacent normal tissues(A) The protein levels of SCAMP3 in HCC (T) and adjacent normal tissues (N) were determined by western blotting in 100 paired samples. SCAMP3 expression was significantly increased in HCC (T) compared with the adjacent normal tissues (N). Representative images of eight paired samples were shown. GAPDH was used as a control. (B) The bar chart shows the ratio of SCAMP3 protein to GAPDH by densitometry, showing a significant higher expression of SCAMP3 in HCC tissues than in the adjacent normal tissues. The data are mean ± SEM of three independent experiments (*P < 0.01, HCC tissues to adjacent normal tissues).

Fig 5: Immunohistochemical analysis of SCAMP3 expression in HCC and adjacent normal tissuesParaffin-embedded HCC tissue sections were stained with antibodies against SCAMP3 and counterstained with hematoxylin. (A, B) Low expression of SCAMP3 in the adjacent normal tissues (SP, A, ×100; B, ×400). (C, D) High SCAMP3 expression in HCC Specimen (SP, C, ×100; D, ×400).