Article Figures & Data
Figures
- FIG 1
Viral genomes are not detected after ex vivo culture of HCMV-positive GBM tumors. (A) DNA isolated from snap-frozen GBM tumors was used as the template for PCR amplification of the HCMV Us28 gene or cellular actin. PCR products were analyzed by agarose gel electrophoresis with ethidium bromide staining. HCMV TB40/E-infected fibroblasts were used as a positive control (+). Mock-infected fibroblasts were used as a negative control. (B) GBM 112 cells were grown in monolayer culture for the indicated number of passages (passage 0 [p0] to passage 3 [p3]) and analyzed as described above. (C) GBM 112, GBM 114, and GBM 120 cells were grown as spheres for the indicated number of passages and analyzed as described above. Images are representative of triplicate PCR experiments.
- FIG 2
Primary GBM cells are permissive to HCMV in vitro. (A) GBM 112 and GBM 114 cells were infected with HCMV TB40/E-mCherry at the indicated MOI for 24 h and then analyzed by flow cytometry for mCherry expression (brown histograms). Mock-infected cells (gray histogram) served as a control. The image is representative of three biological replicates. (B) The average percentage of mCherry-positive (HCMV-infected) cells at 1 day (D1) or 7 days (D7) postinfection for three biological replicates of the experiment presented in panel A are presented. Error bars represent the standard errors of the means for the three biological replicates. (C) GBM 112 spheres infected for 5 days at a MOI of 1.0 with HCMV TB40/E-mCherry (red) and counterstained with 4′,6′-diamidino-2-phenylindole (DAPI) (blue) were imaged by confocal fluorescence microscopy. (D) GBM 112 spheres infected for 7 days at a MOI of 1.0 with HCMV TB40/E-mCherry (red) were imaged by conventional fluorescence microscopy. (Left) Bright-field image; (center) dark-field fluorescence image; (right) bright-field fluorescence image.
- FIG 3
Primary GBM cells support productive HCMV replication in vitro. (A) RNA isolated from GBM 112 and GBM 114 cells infected at the indicated MOI with HCMV TB40/E-mCherry for 72 h was analyzed by RT-PCR for the indicated genes. Mock-infected cells (M) were used as the control. The image is representative of three biological replicates. (B) Supernatants collected at the indicated time (in hours postinfection [hpi]) from GBM 112 and GBM 114 cells infected with HCMV TB40/E-mCherry at the indicated MOI were subjected to a standard plaque assay. Duplicate biological assays were performed. (C) Progeny virus from cell-free or cell-associated samples of GBM 112 or GBM 114 cells infected at a MOI of 1.0 for 144 h were quantitated by a plaque assay. Thee biological replicate experiments were performed and analyzed by Student’s t test for statistical significance. **, P < 0.01; ***, P < 0.001.
- FIG 4
GBM cells infected in vitro with HCMV divide less often than uninfected cells do. (A) GBM 112 cells mock infected or infected at the indicated MOI with HCMV TB40/E-mCherry were grown as spheres, and viable cells were counted at the indicated time (in hours postinfection [hpi]). Averages of three biological replicates are plotted. Values that are significantly different (P < 0.001 by Student’s t test) are indicated (***). (B) Mock-infected or HCMV TB40/E-mCherry-infected GBM cells were loaded with CFSE and then cultured as spheres for 7 days at which time mCherry and CFSE fluorescence of viable cells were quantitated by flow cytometry. For infected cells, the mCherry-negative (mCherry-) and mCherry-positive (mCherry+) populations are shown. The graphs for GBM 112 and GBM 114 cells are representative of three biological replicates. (C) The number of cells within bins representing 1/5th log10 windows is plotted versus the upper limit of the CFSE window for three biological replicates of GBM 112 and GBM 114 cells treated as described above for panel B and one biological replicate of GBM 121 cells. Values that are significantly different by Student’s t test are indicated by asterisks as follows: **, P < 0.01; *, P < 0.05. (D) Cells within the gate representing the top 5% of CFSE staining and mock-infected cells (not shown) were quantified for GBM 112, GBM 114, and GBM 121 cells infected with HCMV at the indicated MOI for 7 days. Averages are plotted with error bars representing the standard errors of the means for biological triplicate samples where appropriate.
- FIG 5
GBM cells infected in vitro with HCMV grow as spheres. GBM 112 and GBM 114 cells infected with HCMV TB40/E-mCherry at the indicated MOI were grown as spheres. At the indicated times, images representing randomly selected (presumably different) spheres were captured. Mock-infected cells served as a control.
- FIG 6
GBM cells infected in vitro with HCMV show increased sphere-forming ability. (A) GBM 112 and GBM 114 cells infected with HCMV TB40/E-mCherry at the indicated MOI were subjected to a sphere formation assay as described in Materials and Methods. Mock-infected cells were used as the control. The average percentages of sphere-forming cells from three biological replicates are plotted with error bars representing the standard errors of the means. ***, P < 0.001; **, P < 0.01. (B) A serial sphere assay (passage 1 [P1] and passage 2 [P2]) was conducted with GBM 114 cells infected with HCMV TB40/E-mCherry at the indicated MOI, analyzed, and displayed as in panel A. Serially passaged mock-infected cells were used as the control. ***, P < 0.001; NS, not significant by Student’s t test.
- FIG 7
GBM 112 and GBM 144 cells infected in vitro with HCMV resist growth inhibition by temozolomide. (A) Sphere formation assays were conducted as described in the legend to Fig. 6 in the presence of carrier (DMSO [D]) or temozolomide (T) where indicated. The average percentages of sphere-forming cells from three biological replicates of GBM 112 and GBM 144 cells (mock infected or infected with HCMV TB40/E) are plotted with error bars representing the standard errors of the means. ***, P < 0.001; **, P < 0.01; NS, not significant by Student’s t test. (B) Sphere formation assays were conducted as described above with the addition of ganciclovir (G) where indicated. The average percentages of sphere-forming cells from three biological replicates from GBM 112 cells (mock infected or infected with HCMV TB40/E) are plotted with error bars representing the standard errors of the means. ***, P < 0.001; **, P < 0.01; *, P < 0.05; NS, not significant by Student’s t test.
