MB cell lines display different levels of MiR-34a induction, which correlate with sensitivity to chemotherapeutic drugs.

<p>(<b>A, B</b>) D283-MED, MHH-Med1 and MEB-Med8A cells were treated with [20 µM] etoposide for indicated time points and levels of miR-34a (A) and Mdm2 mRNA (<b>B</b>) were assessed by real time qPCR. Results were normalised to cyclophilin A and fold changes relative to the untreated control. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108514#pone.0108514.s001" target="_blank">Fig. S1A</a> for a DMSO control. (<b>C</b>) D283-MED cells were transfected with siRNA directed to p53 or with non-specific siRNA as a negative control for 48 hours prior to etoposide treatment. The levels of miR-34a were assessed by qPCR as in (A). Data shown are the mean ± S.E.M of three independent experiments. (A–C) Kruskal-Wallis ANOVA test was performed (*indicates p<0.05). (<b>D</b>–<b>F</b>) Cell viability of D283-MED, MHH-Med1 and MEB-Med8A was measured by MTS assay upon treatment at indicated time points. (<b>D</b>) Etoposide [20 µM] (<b>E</b>) cisplatin [5 µM] (<b>F</b>) methrotrexate [5 µM]. The percentages of viable cells were relative to the untreated control. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108514#pone.0108514.s001" target="_blank">Fig. S1B</a> for a vehicle DMSO control on cell death. Data shown are the mean ± S.E.M of three independent experiments.</p

Expression of WT p53 does not restore p53 activity in MEB-Med8A cells.

<p>(<b>A–C</b>) D283-MED cells were co-transfected with p53-dsRedXP and MDM2-YFP and imaged using time lapse confocal microscopy. The time of etoposide stimulation is represented by the vertical dotted line. The level of p53 and MDM2 were assessed by measuring fluorescence intensity in single cells over time, which were normalised to the baseline fluorescence measured prior to etoposide addition. (B, C): Example of 5 single cell traces and the average fluorescent intensity (red line) of all cells are shown (N = 2, n = 33). (<b>D–F</b>) MEB-Med8A cells were co-transfected and imaged as in (A–C). (E–F): Example of 5 single cell traces and the average fluorescent intensity (red line) of all cells are shown (N = 2, n = 22). (<b>G</b>) A stack column showing the percentage of MB cells with p53 or MDM2 expression above threshold level upon etoposide treatment. Threshold was calculated as average intensity of untreated control +2 SD. D283 cells (N = 2, n = 33); Med8 cells (N = 2, n = 22).</p

miR-34a-mimic induces cell death in p53 mutated MB and GBM cells.

<p>(<b>A</b>) D283-MED, MEB-Med8A MB, U87MG and T98G cells were transfected with [100 nM] miR-34a mimics or non-specific mimic miRIDIAN control and expressed for 72 hours prior to cell viability assessment by MTS assay. Cell viability was normalised to cells transfected with the non-specific mimic control. Data shown are the mean ± S.E.M of four independent experiments. One-way ANOVA followed by Bonferroni test was performed (*indicates p<0.05). (<b>B</b>) D283-MED and MEB-Med8A MB were transfected with [50 nM] or [100 nM] of miR-34a mimic or [50 nM] of non-specific miRIDIAN control for 48 h. Expression levels of SIRT1, c-Myc and Bcl-2 were assessed by western-blot. (<b>C</b>) 2 gels form independent experiments were quantified by densitometry analysis. The plot shown is the result of the quantification relative to actin levels and normalised to cells transfected with the non-specific miRIDIAN control ± sd. (<b>D</b>) D283-MED and MEB-Med8A MB were treated with [20 µM] etoposide for indicated time points. Expression levels of SIRT1 and c-Myc were assessed by western-blot. (<b>E</b>) 2 gels form independent experiments were quantified by densitometry analysis. The graph shown is the result of the quantification normalised to untreated cells at t0 ± sd.</p

miR-34a expression correlates with down-regulation of SIRT1.

<p>(<b>A</b>) mRNA levels of SIRT1 and miR-34a were measured by qPCR upon [20 µM] etoposide treatment at indicated time points in D283-MED cells. (<b>B</b>) SIRT1 protein levels upon [20 µM] etoposide treatment were assessed by western blot. Band density was quantified by densitometry analysis. The blot shown is representative of four independent experiments. (<b>C</b>) SIRT1 levels and localisation were detected by immunofluorescence. Quantification was quantified by densitometry analysis (AQM Advance 6 imaging software) (<b>D</b>) D283-MED and MEB-Med8A cells were treated with [100 mM] nicotinamide for indicated time points. The percentage of cell viability was measured by MTS assay and normalised to the untreated control. Data shown are the mean ± S.E.M of three independent experiments. One-way ANOVA followed by Bonferroni test was performed (*indicates p<0.05). (<b>E, F</b>) Dose response of MB cells treated with nicotinamide. Cells were treated with nicotinamide at indicated concentrations and times and cell viability was measured by MTS assay. (<b>E</b>) D283-MED cells and (<b>F</b>) MEB-Med8A cells. Error bars shown are SD of 6 replicates.</p

p53 activation is impaired in MEB-Med8A cells.

<p>(<b>A</b>) D283-MED cells were treated with [20 µM] etoposide for indicated times and the p53 protein levels were measured by western blot. (<b>B</b>) MEB-Med8A cells were treated with [20 µM] etoposide for indicated times and p53 protein levels were measured by western blot. 2 gels from independent experiments were quantified by densitometry analysis (AQM Advance 6 imaging software, Kinetic Imaging Ltd). The plot shown is the result of the quantification relative to cyclophilin A levels and normalised to t0 untreated control ± sd for each cell line.</p

QDs do not affect the expression of lineage-specific markers in mouse KSCs.

<p><b>A</b>) QPCR analysis of the KSC marker, <i>Wt1</i> mRNA, in unlabelled (QD⁻) and labelled KSCs (QD⁺) after 2 days in culture did not show any significant difference in expression levels. <b>B</b>) Immunofluorescent staining for Wt1 protein (green) showed no difference in expression between control and QD-labelled cells. Nuclei are stained with DAPI (blue). Scale bar −50 µm. <b>C</b>) QPCR analysis of the podocyte-specific marker, <i>synaptopodin</i>, mRNA in unlabelled (QD⁻) and labelled KSCs (QD⁺) after 2 days in culture did not show any significant difference in expression levels. <b>D</b>) Immunostaining for synaptopodin (green) showed nuclear and membrane localisation in both unlabelled (QD⁻) and labelled (QD⁺) podocyte-like cells (yellow arrow). Nuclei are stained with DAPI (blue). Scale bar −50 µm. The reference gene used for QPCR was <i>Gapdh</i>; n = 3 for each experiment.</p

Cell viability and population growth following QD labelling.

<p><b>A</b>) KSC and ESC viability measured by trypan blue exclusion from QD-labelled (QD⁺ - black bar) and unlabelled cells (QD⁻ - grey bar) immediately after QD labelling. <b>B</b>) Population growth curves for unlabelled control ESCs (ESC QD⁻) and QD-labelled ESCs (ESC QD⁺). <b>C</b>) Population growth curves for unlabelled control KSCs (KSC QD⁻) and QD-labelled KSCs (KSC QD⁺); n = 3 for each experiment.</p

QDs do not affect the expression of pluripotency and lineage-specific markers in mouse ESCs.

<p><b>A</b>) QPCR analysis of <i>Oct4</i> mRNA level in unlabelled (ESC QD⁻) and labelled (ESC QD⁺) after 1 and 3 days in culture. <b>B</b>) Immunofluorescent staining for Oct4 protein in both ESC QD⁻ and ESC QD⁺ cells showed nuclear localisation of Oct4 (green); white arrows in lower panel indicate Oct4⁺ QD⁺ cells. Nuclei are stained with DAPI (blue). Scale bar −50 µm. <b>C</b>) Top panel shows that following 4 days of culture under conditions that promote the differentiation of extra-embryonic endoderm, EBs derived from both control (QD⁻) and labelled (QD⁺) ESCs developed a thick layer of extra-embryonic endoderm at the EB periphery. Bottom panel shows that following 4 days of culture under conditions that promote mesoderm differentiation, EBs derived from both QD⁻ and QD⁺ ESCs developed a thin layer of outer extra-embryonic endoderm. Scale bar −100 µm. Images in the right-hand panel show the presence of QDs in 4 day EBs. <b>D</b>) QDs did not affect the size of the EBs under both endoderm- and mesoderm-promoting conditions. <b>E</b>) qPCR analysis of the endoderm-specific gene, <i>Gata6</i> mRNA, the mesoderm-specific gene, <i>brachyury</i> (Bra) and the ectoderm specific gene, <i>Pax6</i>, showed no significant difference in expression levels between EBs generated from QD⁻ ESC and those generated from QD⁺ ESC. The reference gene used for qPCR was <i>Gapdh</i>; n = 3 for each experiment.</p

QDs released following cell death are not readily transferred to neighbouring cells.

<p>(<b>A, B</b>) Phase contrast (A) and fluorescent (B) image of QD-labelled KSC following treatment with DMSO. (<b>C, D</b>) Flow cytometric analysis showed that relatively few QDs released by dead cells were taken up by GFP-labelled KSC; QD from ESCs showed an average uptake of 4.4+/−3.3% (n = 3) (C) and QDs from KSC, 3.4+/−1.2% (n = 3) (D). Scale bars, 20 µm.</p

Primary and secondary antibodies.

<p>Primary and secondary antibodies.</p