Supplementary MaterialsAdditional material. that upon combined treatment with cyclophosphamide and dsDNA, engrafted material loses its tumor-initiating properties which we attribute to the removal of tumor-initiating stem cell subpopulation or loss of its tumorigenic potential. DNA and TAMRA-dUTP precursor by ascites form of Krebs-2 tumor cells. Notably, ascites cells fail to incorporate TAMRA-dUTP. Bar corresponds to 50 m. (B) Same as above, zoom-in, bars correspond to 10 m. Top and side views of a TAMRA-positive cell are shown on the bottom right panel. (C) Fluorescence analysis of total human TAMRA-labeled DNA fragments (0.2C6 kb) internalized by ascites Krebs-2 tumor cells. Bars correspond to 10 m. (D) Molecular analysis of dsDNA internalization by Krebs-2 ascites cells. Upper panel: P32-labeled PCR-amplified GFP fragment was used to directly monitor DNA internalization by ascites cells (probe size is usually shown around the *GFP lane), M, DNA excess weight marker; CP+DNA, Krebs-2 ascites cells collected 18 h post CP injection and incubated with labeled DNA (for 1, 2, 4 and 8 h); DNA, Krebs-2 ascites cells from CP-untreated mice and incubated with labeled DNA (for 1, 2, 4 and 8 h); MC, bone marrow cells from an intact mouse incubated with labeled DNA (for 1, 2, 4 and 8 h). Lower panel: Non-labeled linearized pEGFP-N1/HindIII DNA was used to detect internalization of exogenous DNA by cells. Internalization was visualized by Southern blot using 32P-labeled GFP DNA as a probe. Treatments and collection timepoints are the same as around the upper panel. Southern blot and gel images are shown. Bone marrow cells were used as a positive control, where DNA internalization is usually well-documented.15 (E) FACS profiles showing dsDNA internalization upon increasing concentration of the labeled substrate in the medium. Graph summarizing FACS data are shown below. (F) Circulation cytometry analysis of dynamics of fragment DNA by human glioma cells. (A) Phase-contrast image of adherent cell portion Leflunomide and neurospheres from human glioblastoma cell culture. (B) Fluorescence microscopy analysis of TAMRA-labeled DNA internalization by glioblastoma cells. Background images show the magnitude of non-specific autofluorescence. Notably, labeled DNA probe displays specific nuclear localization in the cells from neurosphere portion, and only a single specific positive transmission was observed across all adherent cells analyzed. (C) Cytofluorescence analysis of TAMRA-labeled DNA in freely-floating neurosphere cell cultures. Several neurospheres are shown. Bottom image represents cytospinned neurospheres stained with DAPI. (D) 3D reconstituted image of a neurosphere with cells internalizing TAMRA-labeled dsDNA (arrowheads). (E) Visualization of GFP expression in neurospheres that have internalized pEGFP-N1 plasmid. As a control, we provide the fluorescence image of a neurosphere (1) and dying glioma cells (2) neither of which were incubated with plasmid DNA. Unless specified otherwise, bars correspond to 50 m. In the beginning we tested the ability Rabbit Polyclonal to DNL3 of cultured human glioma cells to internalize extracellular dsDNA fragments using dissociated adherent cells and neurospheres. Specifically, we directly counted the numbers of TAMRA-positive cells using fluorescence microscopy. The cells from adherent portion failed to internalize TAMRA-labeled dsDNA fragments. In contrast, Leflunomide 7.8% of cells from dissociated neurospheres displayed TAMRA labeling (Fig.?3B). Next, we proceeded to quantify DNA internalization by whole neurospheres and to quantify engraftment efficiency of adherent cell and neurosphere subpopulations in NOD/SKID mice. Our time-lapse imaging experiments showed that neurosphere cells actively internalized TAMRA-labeled dsDNA and became saturated within 1 h. Physique?3C and D shows a panel of neurospheres and a 3D reconstruction, with TAMRA-positive cells clearly present within the neurospheres. We performed direct quantification of TAMRA-dsDNA internalization in squashed preparations of DAPI-stained neurospheres (7 neurospheres altogether), which indicated that about 20% of neurosphere cells were capable of internalizing dsDNA. Our experiments have an obvious translational application, as exogenous dsDNA internalization by human glioma cells may be considered as a encouraging therapeutic target. So, we explored the ability of neurosphere cells to internalize extracellular dsDNA in a form of supercoiled plasmid DNA. We used pEGFP-N1 plasmid for this purpose, as it was shown to transiently transfect eukaryotic cells and produce a fluorescent protein Leflunomide GFP. The results of this analysis are summarized in Physique?3E. Some isolated cells as well cells within neurospheres were GFP-positive. When total DNA from glioma cells incubated with pEGFP-N1 was transformed in qualified DNA. Bars correspond to 10 m (1 and 2) and 50 m (3C5); 6, 3D reconstruction of the tumor fragment. Red dots correspond to individual cells or clusters of cells. To characterize the neurosphere-derived.