Figure 1.
Salivary RNAs reside within salivary ELMs.
(a) Representative electron microscopy images of salivary ELMs, and anti-CD63 immunogold-labeled ELMs in human saliva. (b) Western blot analysis of salivary ELMs using antibodies against human CD63 and GAPDH. Lane 1 to 7 are protein extracts of the salivary ELM pellets obtained from seven donors using Exoquick precipitation. (c) Ribogreen RNA quantitation from seven donors. Error bars represent means ± SD. (d) Representiave electron microscopy images of salivary ELMs and salivary ELMs treated with Tx. (e) RT-qPCR results of three reference genes from the following samples: cell-free saliva; salivary ELMs; salivary ELMs treated with RNase; salivary ELMs treated with Tx and RNase and ELM-depleted saliva. Data are expressed as means ± SD. *P<0.05, statistically significant difference from the saliva group.
Figure 2.
Establishment of a human lung cancer xenograft mouse model.
To generate our xenograft lung cancer mouse model, H460 human lung cancer cells that stably express hCD63-GFP were orthotopically injected into male athymic BALB/c nude mice. CD63 immunolabeled (a, red) and GFP-postive (b, green) H460 CD63-GFP cells are shown. (c) Merge image of CD63, GFP, and DAPI staining. (d) Anti-CD63 and anti-GFP Western blot of ELMs and cell lysates from H460 CD63-GFP and H460 cells. (e) Electron microscopy images of anti-CD63-labeled and anti-GFP-labeled ELMs isolated from the conditioned medium of H460 CD63-GFP cells. Scale bar = 100 nm. (f, g) Nude mice were intrapleurally injected with 1×106 H460 hCD63-GFP cells. H&E staining of pulmonary tumor tissue shows (f) tumor nodule attached to the pleural surface and (g) tumor nodule in the lung parenchyma.
Figure 3.
Detection of human GAPDH mRNA in tumor-bearing mice.
(a) Illustration of anti-hCD63-coated magnetic beads used to isolate hCD63-positive ELMs from saliva, and blood. (b) This native PAGE analysis depicts the 72-bp nested RT-PCR products for human GAPDH from H460-hCD63-GFP cells, LLC1 cells, and (c) the blood and saliva of 15 H460-hCD63-GFP tumor-bearing or control mice.
Table 1.
Detection of human GAPDH mRNA in saliva and blood from mice bearing hCD63-GFP tumors.
Figure 4.
hCD63-GFP-positive ELMs in saliva and blood from tumor-bearing mice.
(a) Acetylcholinesterase quantification of ELMs from the conditioned media of H460-hCD63-GFP cells treated with 1 µmol or 10 µmol DMA for 48 h. (b) Trypan blue exclusion assay of H460-hCD63-GFP cells after 48 h DMA treatment. (c) Illustration of the measurement of hCD63-positive ELMs in saliva and blood using EFIRM technology, allowing the detection of hCD63-GFP positive ELMs by electrochemical sensors. Samples of (d) blood and (e) saliva from control or tumor-bearing mice treated with PBS or DMA for 1 week and assayed for hCD63-GFP-positive ELMs using EFIRM technology. N = 9–11 per group; *P<0.05, statistically significant differences.