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Cancers 2018, 10, x FOR PEER REVIEW 9 of 15 Cancers 2018, 10, 144 9 of 15 the combination of OAdmCherry and TMZ has a potent inhibitory effect upon colony formation in murine TNBC cells. Figure 6. Combined therapy increases viral replication and strongly inhibits clonogenic survival in Figure 6. Combined therapy increases viral replication and strongly inhibits clonogenic survival mouse triple-negative breast cancer (TNBC) cells: (A) 4T1 cells were infected with oncolytic in mouse triple-negative breast cancer (TNBC) cells: (A) 4T1 cells were infected with oncolytic adenovirus mCherry (OAdmCherry) or adenovirus expressing green fluorescent protein (AdGFP) at adenovirus mCherry (OAdmCherry) or adenovirus expressing green fluorescent protein (AdGFP) at different multiplicity of infection (MOI) concentrations for 72 h. Crystal violet staining was used to different multiplicity of infection (MOI) concentrations for 72 h. Crystal violet staining was used to evaluate cytopathic effect (CPE). A representative staining of three independent experiments is evaluate cytopathic effect (CPE). A representative staining of three independent experiments is shown. shown. (B) Relative cell survival was calculated by measuring the absorbance of solubilized dye at (B) Relative cell survival was calculated by measuring the absorbance of solubilized dye at 590 nm. 590 nm. (C) 4T1 cells were treated with TMZ at different concentrations for 72 h. Cell survival was (C) 4T1 cells were treated with TMZ at different concentrations for 72 h. Cell survival was calculated by calculated by MTT assay. Results represent the mean of three repeated measurements ± standard MTT assay. Results represent the mean of three repeated measurements ± standard deviation (SD; error deviation (SD; error bars) (* p < 0.05). (D) 4T1 cells were infected with OAdmCherry at a MOI bars) (* p < 0.05). (D) 4T1 cells were infected with OAdmCherry at a MOI concentration of 10 alone or in concentration of 10 alone or in combination with TMZ or vehicle dimethyl sulfoxide (DMSO). combination with TMZ or vehicle dimethyl sulfoxide (DMSO). Expression of mCherry was evaluated Expression of mCherry was evaluated by fluorescence microscopy. Scale: 200 μm. (E) Percentage of by fluorescence microscopy. Scale: 200 μm. (E) Percentage of mCherry-positive cells calculated relative mCherry-positive cells calculated relative to number of cells in the field. Results represent the mean to number of cells in the field. Results represent the mean of three repeated measurements ± standard of three repeated measurements ± standard deviation (SD; error bars) (* p < 0.05). (F) Expression of deviation (SD; error bars) (* p < 0.05). (F) Expression of (Ad) E1A was evaluated 72 h post treatment (Ad) E1A was evaluated 72 h post treatment by Western blot assay. Actin was used as a loading by Western blot assay. Actin was used as a loading control. A representative assay is shown from control. A representative assay is shown from three performed. (G) 4T1 cells were infected with three performed. (G) 4T1 cells were infected with OAdmCherry alone or in combination with TMZ OAdmCherry alone or in combination with TMZ or vehicle DMSO. Crystal violet staining was used or vehicle DMSO. Crystal violet staining was used to evaluate clonogenic survival. (H) Percentage of cell survival calculated relative to the untreated control. Results represent the mean of three repeated measurements ± standard deviation (SD; error bars) (* p < 0.05).PDF Image | Temozolomide Enhances Triple-Negative Breast Cancer Virotherapy
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