Multiple cross-spectral function of myxoma virus proteins (10 sequences alignment).

<p>Multiple cross-spectral function of myxoma virus proteins (10 sequences alignment).</p

Cellular viability of mammalian cell lines treated with a single dose of different concentrations of the RRM peptides up to 72 h after treatment.

<p><b>A.</b> CHO, <b>B.</b> B16F0 and <b>C.</b> 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 (<i>p</i><0.05).</p

CLSM micrographs for human squamous carcinoma COLO 16 cell line.

<p>Cell cultures were treated with 50 ng/ml, 100 ng/ml and 200 ng/ml of RRM-MV for 3 h in <b>A</b>, <b>B</b>, and <b>C</b> respectively. Cell culture in <b>D</b> was treated with 200 ng/ml of RRM-C, while cell cultures in <b>E</b> 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.</p

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).

<p>After 3 h incubation with DMEM only in <b>A</b> (blank), 3 h incubation with 800 ng/ml RRM-C in <b>B</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 <b>C</b> when compared with A and B. Longer treatment periods 6 h; 9 h; and 18 h in (<b>D–F</b> respectively) with increased levels of necrosis and cellular detachment when B16F0 cell cultures were treated with (800 ng/ml) of RRM-MV. (200× magnification).</p

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.

<p>Mouse skin fibroblasts in <b>A</b>; mouse macrophages J774 in <b>B</b>; and CHO in <b>C</b>. 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.</p

Cytotoxic effect of RRM peptide analogues on normal and cancer cells measured by LDH assay.

<p>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 <b>*</b> (when compared to control treated cells) and <b>+</b> (when compared to untreated cells) at a significant level of <i>p</i><0.05.</p

The effect of RRM peptide treatment on total Akt and p- Akt in B16F0 cell line.

<p><b>A.</b> Western blots for total Akt (pan) Rabbit MAB in <b>I</b> and p-Akt (Thr308) Rabbit MAB in <b>II</b> without Akt inhibitor. <b>B.</b> Western blots for B16F0 cells treated with 50 µM LY294002 (PI3 kinase inhibitor) prior to immunoblotting with total Akt rabbit MAB in <b>III</b> and p-Akt (Thr308) rabbit MAB in <b>IV</b>. In <b>A</b>, 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>B</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.</p