Fig 1.
Reporter cells subjected to exosomes or conditioned culture medium from UV-exposed bystander cells (UV emitted from beta-irradiated cells).
(A) Surviving fraction of HCT116 p53 +/+ cells cultured in UV-exposed ICCM or CCCM. Error bars represent SEM for 18 replicates (3 replicates for each of 6 independent experiments) for the UV-ICCM treatment and the CCCM control, and 9 replicates (3 replicates for 3 independent experiments) for the UV-exposed medium (no cell) control. (B) Surviving fraction of HCT116 p53 +/+ cells cultured in exosomes extracted from UV-exposed ICCM or CCCM. Error bars represent SEM for 18 replicates (3 replicates for each of 6 independent experiments) for the UV-ICCM exosome treatment and CCCM exosome control, and 9 replicates (3 replicates for 3 independent experiments) for the no cell control & medium only control. Letters (a,b,c) indicate significant differences between samples as assessed by means of 1-way ANOVA, 95% confidence level.
Fig 2.
Fluorescence of JC-1 dye incubated with HCT116 p53 +/+ reporter cells which received.
(A) exosomes extracted from ICCM treated with cell-emitted UV biophotons and (B) exosomes extracted from CCCM which did not receive UV biophoton irradiation. Fluorescence microscopy images were acquired using an Olympus IX81 microscope and Image Pro AMS 5.1 software. (C) Mitochondrial membrane potential observed in HCT116 p53+/+ cells following the receipt of exosome fractions extracted from UV-exposed bystander cells. The UV was emitted from directly-irradiated cells that were exposed to 0.5 Gy 3H β-radiation. Error bars represent SEM for a total of 18 replicates (6 replicates for each of 3 independent experiments). Fluorescence ratios were normalized to the DMSO negative control.
Fig 3.
Reporter cells subjected to RNase-treated ICCM or exosomes derived from UV-exposed bystander cells.
(A) Clonogenic survival of HCT116 p53 +/+ reporter cells receiving RNase-treated UV-ICCM, UV-ICCM, or CCCM. Error bars represent SEM for a total of 18 replicates (3 replicates for each of 6 independent experiments) for the 0.5 Gy positive control and 0 Gy negative control, and 9 replicates (3 replicates for 3 independent experiments) for the RNase-treated 0.5 Gy group. (B) Clonogenic survival of HCT116 p53 +/+ reporter cells following treatment of the reporter cells with RNase-treated UV-exposed exosomes, UV-exposed exosome fractions (no RNase treatment), or non-exposed exosome fractions. Error bars represent SEM for a total of 18 replicates (3 replicates for each of 6 independent experiments) for the 0.5 Gy positive control and 0 Gy negative control, and 9 replicates (3 replicates for 3 independent experiments) for the RNase-treated 0.5 Gy group. (C) Mitochondrial membrane potential (assessed via the incubation of cells with JC-1 mitochondrial potential dye) of HCT116 p53 +/+ reporter cells receiving RNase-treated or non-RNase-treated exosome isolates extracted from ICCM or CCCM. Error bars represent standard error of the mean for 6 replicates tested for each of three independent experiments (18 replicates total). Letters (a,b,c) represent significant differences between treatments as assessed by 1-way analysis of variance; post-hoc testing assessed using Tukey’s HSD test at the 95% confidence level.
Fig 4.
Protein bands acquired using western blots for expression of.
(A) CD63 (glycosylated form: 30–70 kDa, non-glycosylated form: 25 kDa), (B) TSG101 (49 kDa), and (C) Actin (42 kDa). Lane 1: positive control (10 μg protein from HepG2 whole cell lysate for CD63 and actin antibodies; 10 μg protein from HeLa whole cell lysate for TSG101 antibody). Lane 2: exosomes extracted from HCT116 p53 +/+ CCCM. Lane 3: HCT116 p53 +/+ whole cell lysate not exposed to UV. Lane 4: exosomes extracted from HCT116 p53 +/+ UV-ICCM. Lane 5: HCT116 p53 +/+ whole cell lysate exposed to UV. All lanes contain 10 μg protein each. The lack of actin expression demonstrated by lanes 2 and 4 indicate the absence of actin in exosome samples. Because actin is not required for exosome transport, the absence of actin in exosome samples is expected and indicates a lack of contamination by cellular debris in the exosome isolates.
Fig 5.
Transmission electron microscopy images illustrating the exosomes that were extracted from HCT116 p53 +/+ cells via ultracentrifugation.
Exosomes are indicated by arrowheads. Scale bars in each of the four panels represent 100 nm. (A) Exosomes extracted from UV-exposed cells, direct magnification: 120 000x. (B) Exosomes extracted from non-UV-exposed control cells, direct magnification: 100 000x. (C) Exosomes from UV-exposed cells, direct magnification: 140 000x. (D) Exosome from UV-exposed cells, direct magnification: 160 000x.