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. 2023 Mar 17;12(3):408–426. doi: 10.21037/tlcr-22-571

Figure 1.

Figure 1

CKAP4 is secreted with SEVs from lung cancer cells. (A) SEVs prepared from the CM, cell lysates, and precipitated PM proteins of NSCLC cells were probed with indicated antibodies. (B) SEVs prepared from the CM of A549 cells using MagCapture were subjected to discontinuous sucrose gradient analysis. CD81 and TSG101 are exosome marker proteins. (C) SEVs were prepared from the CM of WT-A549 or A549/DKK1-FLAG cells transfected with control (scramble) or DKK1 siRNAs. (D,F) SEVs were prepared from the CM of A549 cells transfected with control or indicated siRNAs. Cells were biotinylated and PM proteins precipitated. (E) SEVs were prepared from the CM of A549 cells treated with or without 25 µM MDC for 48 h. (G) SEVs prepared from the CM of Calu-1 cells stably expressing control shRNA, DKK1 shRNA, and/or DKK3 shRNAs. (H,I) SEVs were prepared from the CM of WT-NCI-H292 and NCI-H292/CKAP4-HA cells transfected with control or DKK1 siRNAs. (J) Serum CKAP4 levels in immunodeficient mice implanted with WT-NCI-H292 (n=3) or NCI-H292/CKAP4-HA (n=3) cells were measured using sandwich ELISA. Results are indicated by a box plot. The median is represented with a line, the box represents the 25th–75th percentile, and error bars show the 5th–95th percentile. The band intensities of exosomal CKAP4 (C-G and I) and Clathrin (D) were quantified and expressed as AU (control value in each assay is 1). SEVs, small extracellular vesicles; CM, conditioned medium; PM, plasma membrane; NSCLC, non-small cell lung cancer; WT, wild-type; MDC, monodansyl cadaverine; ELISA, enzyme-linked immunosorbent assay; AU, arbitrary unit.