Figure 2.
AGER is required for exosome uptake of KRASG12D by macrophages. (A) Transmission electron microscopy image of the exosomes isolated from PANC1 cells. (B) nanoparticle tracking analysis for the exosomes isolated from PANC1 cells. (C) Western blot analysis of exosome markers in PANC1 cells. CD63, CD81, PDCD6IP/ALIX, and TSG101 are used as exosome markers, and HSP90B1/GRP9 and CYCS are used as markers of cellular contamination. WCL, whole cell lysate; Exo, exosome. (D) Western blot analysis of KRASG12D, CD63, and PDCD6IP/ALIX expression in isolated exosomes from PDAC cells following treatment with H2O2 (500 µM) in the absence or presence of chloroquine (50 µM) or ferrostatin-1 (1 µM) for 24 h. (E) Western blot analysis of KRASG12D, CD63, and PDCD6IP/ALIX expression in isolated exosomes from wild-type and ATG5-knockdown PDAC cells following treatment with H2O2 (500 µM) for 24 h. (F) Analysis of KRASG12D release in the supernatant in PDAC cells following treatment with H2O2 (500 µM) in the absence or presence of GW4869 (10 µM) for 24 h (n = 3, *P < 0.05 versus H2O2 group). (G) Analysis of KRASG12D release in the supernatant in wild-type and RAB27A-knockdown PANC1 cells following treatment with H2O2 (500 µM) for 24 h (n = 3, *P < 0.05 versus wild-type group). (H) Western blot analysis of indicated protein expression in the successive pellets (2K, 10K, and 100K) of extracellular vesicles isolated from H2O2-treated PANC1 cells. (I) Proteinase protection assay of 100K pellets in the absence or presence of proteinase K (1 mg/mL) or 1% Triton X-100 for 30 min at 37°C. (J) Western blot analysis of KRASG12D and ACTB expression in PBMCMs following treatment with 100 μg/ml exosomes isolated from H2O2 (500 µM, 24 h)-treated PANC1 cells for 6–24 h. (K) Western blot analysis of KRASG12D and ACTB expression in PBMCMs following treatment with 100 μg/ml exosomes isolated from H2O2 (500 µM)-treated PANC1 cells in the absence or presence of AGER Ab (50 µg/ml) and control IgG (50 µg/ml) for 24 h. (L) Western blot analysis of KRASG12D and ACTB expression in wild-type and AGER-knockdown PBMCMs following treatment with 100 μg/ml exosomes isolated from H2O2 (500 µM)-treated PANC1 for 24 h