Fig 2: Sec23a inhibits lung-derived metastasis of melanoma cells.a Representative images of OL-N.C., OL-shSec23a, POL-vector, and POL-Sec23a-OE cells. Scale bars, 200 µm. b, c RT-qPCR and western blot were performed to confirm gene manipulation of Sec23a expression in OL and POL. ***P < 0.001. d, e Representative images and quantification of transwell migration and invasion assay. Scale bars, 60 µm. ***P < 0.001. f, g Representative images and quantification of colony formation assay. *P < 0.05. h Lung-derived metastatic nodules were shown by whole-lung bright, fluorescent and H&E images. Scale bars, 1 mm. i Quantification of lung-derived metastatic nodules. **P < 0.01.

Fig 3: SEC23A is a direct target of miR-1227; (A) qPCR for high confidence miR-1227 target genes involved in vesicle formation from PC3 cells stably expressing miR-1227 (n = 3). (B) Western blot validation of SEC23A reduced expression in PC3, RWPE-2, and U87 cells stably expressing miR-1227. (C) Prostate cancer transcriptome atlas analysis of SEC23A in prostate cancer, showing reduced expression levels of SEC23A in cancer versus benign prostate tissue and in metastatic castration resistant versus primary prostate cancer. (D–F) Luciferase assays from RWPE-2 cells transfected with miR-1227 or control miRNA, plus the SEC23A 3'UTR luciferase construct (D) or the SEC23A 3'UTR luciferase construct with the deletion of 2 different predicted miR-1227 binding sites (E,F) (n = 3); * p < 0.05; ** p < 0.005.

Fig 4: S100A8 transported by SEC23A augments autophagy in melanoma cells.a Quantitative analysis of autophagy-associated secreted protein transported by SEC23A. b Western blot analysis for S100A8 in the conditional medium of OL-N.C., OL-shSec23a, POL-vector, and POL-Sec23a-OE cells. c Western blot analysis for S100A8 in the conditional medium of tumor cells treated with Brefeldin A. d RT-qPCR was performed to confirm S100A8 interference in OL and POL-Sec23a-OE cells. **P < 0.01. e Western blot analysis for S100A8, LC3I/II and p62 in OL, and POL-Sec23a-OE cells with S100A8 interference. f, g Western blot analysis for LC3I, LC3II, and P62 in tumor cells treated with anti-S100A8/A9 antibody or recombinant S100A8/A9 protein dimer. h–j LC3B-positive puncta detected in tumor cells after S100A8 interference, anti-S100A8/A9 antibody treatment, or recombinant S100A8/A9 protein dimer treatment. Scale bars, 20 µm. k–n Detection and quantification of autolysosomes in tumor cells after S100A8 interference, anti-S100A8/A9 antibody treatment or recombinant S100A8/A9 protein dimer treatment. Autolysosomes indicated by arrowheads in TEM images. Scale bars, 5 µm. **P < 0.01, ***P < 0.001. o–q Western blot analysis for BECLIN1 in tumor cells after S100A8 interference, anti-S100A8/A9 antibody treatment or recombinant S100A8/A9 protein dimer treatment. r Co-IP assay to detect interaction between S100A8 and BECLIN1.

Fig 5: Sec23a inhibits the stemness of M14-SE cells via inhibition of autophagy. a Western blot analysis for LC3I/II and P62 in M14-SE cells with Sec23a interference or overexpression. b Representatives of LC3B-positive puncta images were shown in M14-SE cells with Sec23a interference or overexpression, bar = 20 µm. c Transmission electron microscopy of M14-SE cells with Sec23a interference or overexpression. Autolysosomes indicated by arrowheads, bar = 5 µm. d Mean number of detectable autolysosomes in each tumor cell, counted on transmission electron microscopy images. e and f RT-qPCR and Western blot analysis to confirm effective Atg5 interference in M14-SE-shSec23a cells. g Analysis of mRNA expression of Aldh1, CD133, Nanog, Oct4 and Sox2 in M14-SE-shSec23a cells with Atg5 interference. h–j. Clonogenic spheroid formation rate, morphology and average spheroid diameter of M14-SE-shSec23a cells with Atg5 interference in the single-cell cloning assay. k Sserial spheroid formation assay of M14-SE-shSec23a cells with Atg5 interference, bar = 200 µm. l Tumors resected from nude mice inoculated with M14-SE-shSec23a cells with Atg5 interference, bar = 1 cm. (m and n). Tumor volume and weight, **P < 0.01, ***P < 0.001