Fig 2: Putative model describing that metformin suppressed melanoma cell growth and motility through modulating the miR-192-5p/EFEMP1 and miR-584-3p/SCAMP3 axes.

Fig 3: Knockdown of EFEMP1 and SCAMP3 expression influenced the cell invasion ability of melanoma cells. (A) and (C): Migration and invasion ability were examined by Transwell assay in A2058 cells. After knockdown of EFEMP1 and SCAMP3, or scramble control transfection for 24 h, cells were subjected to Transwell assay. The migrating and invading cells were stained with crystal violet solution. (B) and (D): Then, the numbers of migrating or invading cells were quantified by counting three different fields under a phase-contrast microscope. The cell photographs from a representative experiment are presented and the graph data were quantified using Ascent software. Data are reported as colonies compared with control (means ± SD).

Fig 4: Knockdown of EFEMP1 and SCAMP3 expression significantly inhibited melanoma cell growth. (A) and (B): After siRNA transfection, the expression levels of EFEMP1and SCAMP3 were examined through real-time PCR and Western blotting. (C) and (D): Colony formation assay was examined in A2058 cells with EFEMP1 and SCAMP3 knockdown; and the relative colony formation ability was quantified using OD595 nm. (E) and (F): Cell proliferation was assessed in A2058 cells with EFEMP1 and SCAMP3 knockdown using the CellTiter-Glo One solution assay.

Fig 5: Budding of WAEVs is mediated by SCAMP3.(A) SDS-PAGE gel with Coomassie blue staining of proteins from control and M2-WAEVs. About 6 × 109 of EVs were loaded. (B) Volcano plot of proteins in WAEVs. Up-regulated are marked as orange; down-regulated are marked as purple. (C) Western blotting showing increased presence of SCAMP3 in M2-WAEVs. (D) SCAMP3 cosegregates with M2-WW in fractionated EVs. M2-WAEVs were isolated via ultracentrifugation and subject to OptiPrep density gradient ultracentrifugation. (E) SCAMP3 coimmunoprecipitates with M2-WW. HEK293T cells were transfected with M2-WW (FLAG-tagged) along with either wild-type or mutant SCAMP3 (GFP-tagged). Immunoprecipitation (IP) was done using an anti-FLAG antibody conjugated beads. (F and G) Effect of CRISPR-based KO of SCAMP3 on M2-WAEV budding. HEK293T cells stably transduced with lentiviruses containing scrambled gRNA or SCAMP3-targeting gRNAs were transfected with M2-WW. EVs were isolated from the cells and subject to Western blotting (F) or NanoSight particle analysis (G). Data were obtained from triplicates for each condition. n.s., not significant; ***P < 0.001. (H and I) Effect of reexpression of wild-type or mutant SCAMP3 in SCAMP3-KO cells on M2-WAEV budding. HEK293T cells with SCAMP3-KO (KO1) transfected with wild-type or mutant SCAMP3. Both SCAMP3 constructs contain silent mutations in the gRNA targeting region so are resistant to the CRISPR targeting. EVs were isolated from the cells and subject to Western blotting (H) or NanoSight particle analysis (I). Data in the vesicle number analysis were obtained from triplicates for each condition. ***P < 0.001. (J) Effect of siRNA-based knockdown of TSG101 on M2-WAEV budding. EVs were isolated from control or TSG101 knockdown cells and analyzed by Western blotting. (K) Quantification of Western blotting data in (J). ***P < 0.001. (L) NanoSight particle analysis of EVs. Data were obtained from triplicates for each condition. *P < 0.05. (M) A model of WAEV budding.