Figure 1.
Multiple cross-spectral function of myxoma virus proteins (10 sequences alignment).
Figure 2.
CLSM micrographs for apoptosis/necrosis assay with annexin V-Alexa Fluor 488 (green fluorescence) and propidium iodide (red fluorescence) in mouse melanoma cell line (B16F0).
After 3 h incubation with DMEM only in A (blank), 3 h incubation with 800 ng/ml RRM-C in B, and with 800 ng/ml RRM-MV in C. Cytotoxic changes including detachment of confluent layer, apoptotic cells (green) and necrotic cells (red) and are obvious in C when compared with A and B. Longer treatment periods 6 h; 9 h; and 18 h in (D–F respectively) with increased levels of necrosis and cellular detachment when B16F0 cell cultures were treated with (800 ng/ml) of RRM-MV. (200× magnification).
Figure 3.
CLSM micrographs for human squamous carcinoma COLO 16 cell line.
Cell cultures were treated with 50 ng/ml, 100 ng/ml and 200 ng/ml of RRM-MV for 3 h in A, B, and C respectively. Cell culture in D was treated with 200 ng/ml of RRM-C, while cell cultures in E were similarly incubated without any treatment. More necrotic cells and detachment can be seen in B and C as compared with A indicating dose-dependent cytotoxic effect of RRM-MV. No cellular detachment can be seen in the cell culture treated with 200 ng/ml of the negative control RRM-C in D or in the non-treated cell culture in E.
Figure 4.
CLSM micrographs for the apoptosis/necrosis assay in three normal cell lines after 3 h incubation with 800 ng/ml of RRM-MV; or 800 ng/ml of RRM-C.
Mouse skin fibroblasts in A; mouse macrophages J774 in B; and CHO in C. No significant cytotoxic effects (apoptosis, necrosis and cellular detachment) were detected in all cell cultures treated with RRM-MV or RRM-C as compared with the non-treated cell cultures similarly incubated in DMEM, indicating the minimal cytotoxic effect of RRM-MV on the 3 normal cell lines.
Figure 5.
Cytotoxic effect of RRM peptide analogues on normal and cancer cells measured by LDH assay.
Cells (3×105) were incubated for 3 h with control peptide (RRM-C), or with RRM-MV at 400 ng/ml (for COLO16) and 1600 ng/ml (for CHO, J774A.1 and B16-F0). Cells without treatment were similarly incubated for 3 h (blank). Each bar represents mean ± standard errors of 3 separate experiments in triplicate. Data values that are significantly altered (ANOVA and Dunnett's post-hoc analysis) are indicated by * (when compared to control treated cells) and + (when compared to untreated cells) at a significant level of p<0.05.
Figure 6.
Cellular viability of mammalian cell lines treated with a single dose of different concentrations of the RRM peptides up to 72 h after treatment.
A. CHO, B. B16F0 and C. COLO16 cells. Cell cultures (1×105) were incubated for 4 h, 8 h, 16 h, 24 h, 48 h and 72 h with (50 ng/ml, 100 ng/ml, 200 ng/ml, 400 ng/ml and 800 ng/ml) of RRM-MV and 400 ng/ml of RRM-C. A blank (no treatment control) and a positive control (treated with 60% DMSO) were included in all assays. OD was measured at 570 nm after addition of the Prestoblue™ reagent and incubation for 30 min. Cell viability was calculated and is shown relative to that of untreated (blank) sample (set to 100%). Each bar represents mean ± standard errors of 3 separate experiments in triplicate. Data values that are significantly altered (ANOVA and Dunnett's post hoc analysis) when compared to the untreated cells are indicated by the star symbol (p<0.05).
Figure 7.
Cellular viability of mouse melanoma cells B16F0 overtime following a repeated dose of the RRM peptides.
Cell cultures (1×105) were first treated with 50 ng/ml, 100 ng/ml, 200 ng/ml, 400 ng/ml and 800 ng/ml of RRM-MV and 400 ng/ml of RRM-C for 16 h. A second dose of RRM-C (400 ng/ml) or RRM-MV (50 ng/ml, 100 ng/ml, 200 ng/ml, 400 ng/ml and 800 ng/ml) or DMSO (positive control for cell cytotoxicity) was added after the initial 16 h treatment and cell cultures were then incubated further for another 10 h, 24 h and 48 h. OD was measured at 570 nm after addition and incubation with the Prestoblue™ reagent and cell viability was calculated. Cell viability of all treated samples is shown relative to that of untreated (blank) sample (set to 100%). Each bar represents mean ± standard errors of 3 separate experiments in triplicate. Data values that are significantly altered (ANOVA and Dunnett's post hoc analysis) when compared to the untreated cells are indicated by the star symbol (p<0.05).
Figure 8.
The effect of RRM peptide treatment on total Akt and p- Akt in B16F0 cell line.
A. Western blots for total Akt (pan) Rabbit MAB in I and p-Akt (Thr308) Rabbit MAB in II without Akt inhibitor. B. Western blots for B16F0 cells treated with 50 µM LY294002 (PI3 kinase inhibitor) prior to immunoblotting with total Akt rabbit MAB in III and p-Akt (Thr308) rabbit MAB in IV. In A, Cells were grown in DMEM only in lane 1; DMEM and 800 ng/ml RRM-C in lane 2; and DMEM with 800 ng/ml RRM-MV in lane 3. In B, cells were either grown in DMEM in lane 1, or in DMEM with 50 µM LY294002 for 1 h in lane 2. Similar intensities of the 60 kDa immune bands for total Akt and p-Akt in treated and non treated cells in A indicate the lack of effect of the RRM-designed peptides on p-Akt activity as compared with the inhibitory effect of the Akt inhibitor on p-Akt activity in B.