ODC activity in JIMT-1, L56Br-C1, MCF-7 and MCF-10A cells treated with NSpd, Pd-NSpd or Pt-NSpd.

<p>Twenty-four h after seeding of cells (0 h time of treatment in the figure), NSpd, Pd-NSpd or Pt-NSpd was added to give a final concentration of 25 µM. The ODC activity was determined using a radiometric assay. The results are presented as mean values (n = 3 independent samples from one independent experiment) and bars represent ± SD.</p

Additional file 3: Figure S3. of Breast cancer stem cell selectivity of synthetic nanomolar-active salinomycin analogs

Representative cytograms of ALDH assay obtained using flow cytometry. JIMT-1 cells were treated with 50 nM salinomycin or salinomycin analogs for 72 h. SA: salinomycin, 2a: carbamate, 2b: acetate, 2c: carbonate and 3: C1-methyl ester. (DOCX 1678 kb

Effects of NSpd, Pd-NSpd or Pt-NSpd on the uptake of ³H-spermidine in JIMT-1, L56Br-C1, MCF-7 and MCF-10A cells.

<p>The cells were seeded in 12 well plates and incubated for 48 h, whereupon the polyamine analogue or its complexes were added to give the final concentrations shown in the figure. The concentration of ³H-spermidine used was 1 µM. The results are presented as mean values (n = 4 samples from two independent experiments) and bars represent ± SD.</p

Sub-G₁ region and cell cycle phase distribution of JIMT-1, L56Br-C1, MCF-7 and MCF-10A cells treated with NSpd, Pd-NSpd or Pt-NSpd.

<p>Twenty-four h after seeding the cells, NSpd, Pd-NSpd or Pt-NSpd were added to give a final concentration of 100 µM. At 24, 48 and 72 h of treatment, both detached and attached cells were harvested, pooled and fixed in 70% ice-cold ethanol. The nuclei were stained with propidium iodide and the analysis was performed using flow cytometry. The results are presented as mean values (n = 3−6 independent samples from one or two independent experiments) and bars represent ± SD.</p

Intracellular accumulation of Pd-NSpd and Pt-NSpd in L56Br-C1 and MCF-10A cells.

<p>At 72 h of treatment with 25 µM of Pd-NSpd and Pt-NSpd, cells were harvested, pooled and digested in HNO₃. The supernatant was used for analysis of Pd and Pt by ICP-MS and the data used to calculate the intracellular Pd-NSpd and Pt-NSpd concentrations. The results are presented as mean values (n = 3 independent samples from one independent experiment) and bars represent ± SD.</p

Additional file 7: Figure S7. of Breast cancer stem cell selectivity of synthetic nanomolar-active salinomycin analogs

Cell cycle effects of treating with 50 nM salinomycin or the analogs 2a-c for 72 h. (a) Cell cycle phase distribution evaluated using flow cytometry. (b) Representative Western blots used for densitometric scanning to obtain the data in (c) and (d). (c-d) Expression of cyclin A2 and p27, respectively. The columns in (c) and (d) show mean ± SEM for n = 6. * P < 0.05. SA: salinomycin, 2a: carbamate, 2b: acetate, 2c: carbonate and 3: C1-methyl ester. (JPG 737 kb

Dose response effects of NSpd, Pd-NSpd or Pt-NSpd treatment.

<p>The three breast cancer cell lines JIMT-1 (<b>A</b>–<b>C</b>), L56Br-C1 (<b>D</b>–<b>F</b>), MCF-7 (<b>G</b>–<b>I</b>) and the normal-like breast cell line MCF-10A (J–L) were used. Twenty-four h after seeding of cells in 96-well plates, the polyamine analogue and its complexes were added to the final concentrations shown in the figure and the cells were treated for 24, 48 and 72 h, before evaluation using an MTT assay. The results are expressed as % of control (n = 12 independent samples from two independent experiments) with bars representing ± SD.</p

The single cell gel electrophoresis (SCGE) assay was used to evaluate DNA damage in L56Br-C1 cells.

<p>Twenty-four h after seeding of L56Br-C1 cells, NSpd, Pd-NSpd or Pt-NSpd was added to give a final concentration of 25 µM. After 72 h of treatment, cells were harvested for SCGE analysis. The ethidium bromide-stained nucleoids were photographed and then examined using the Comet Score™ Freeware. <b>A</b>. Images of comets obtained by the SCGE assay. DNA damage results in comets with head and tail, whereas undamaged DNA results in a round head. <b>B</b>. Percentage DNA in tail on the x-axis <i>versus</i> tail length on the y-axis for individual cells. <b>C</b>. Tail moment TMOM (%DNA in tail multiplied by tail length) for individual cells. Data were collected from three independent experiments, n = 207 cells. <b>D</b>. Table showing the mean TMOM value of the 10% highest TMOM values <i>i.e.</i> 20 highest values ± SD. *p<0.05 compared to control; ***p<0.001 compared to control.</p

Effects of NSpd, Pd-NSpd or Pt-NSpd treatment on the proliferation of JIMT-1, L56Br-C1, MCF-7 and MCF-10A cells.

<p>Twenty-four h after seeding of cells (0 h time of treatment in the figure), NSpd, Pd-NSpd or Pt-NSpd was added to give a final concentration of 25 µM (<b>A</b>–<b>D</b>) or 100 µM (<b>E</b>–<b>H</b>). Cells were harvested by trypsinization and counted in a hemocytometer. The results are presented as mean values (n = 3−6 independent samples from one or two independent experiments) and bars represent ± SD. When not visible, the bars are covered by the symbols. <b>I</b>–<b>L</b>: Cells were seeded and NSpd, Pd-NSpd or Pt-NSpd was added to the final concentration of 25 µM after 24 h of seeding. After 72 h of treatment, the drug-containing medium was aspirated and drug free culture medium was added. After an additional 72 h of incubation, cells were harvested by trypsinization and counted in a hemocytometer. These 7 days were defined as one treatment cycle. The total recovery time between each treatment was 96 h. The cells were reseeded at the same density as at the previous passage and treated with the same drug for the next treatment cycle. All together this was repeated for 5 treatment cycles. The data are presented as the total amount of cells (mean values (n = 3−6 samples from one or two independent experiments) and bars represent ± SD) that theoretically would have accumulated if all cells had been reseeded with a known cell density after each treatment cycle. When not visible, the bars are covered by the symbols. Please note that the y-axis has different scales for the different cell lines because of different rates of cell proliferation.</p

Additional file 4: Figure S4. of Breast cancer stem cell selectivity of synthetic nanomolar-active salinomycin analogs

Salinomycin treatment decreases the proportion of ALDH+ in a dose dependent manner. JIMT-1 cells were treated with salinomycin for 72 h at the indicated concentrations. The effect on the ALDH+ population was determined using flow cytometry. Data are represented as mean ± SEM for n = 3. (DOCX 38 kb