c-Myc knockdown abolishes xenograft tumor formation by glioma cancer stem cells.

<p>(A) T3359 CD133+ cells were infected and selected as described. After selection, cells were injected into brains of athymic BALB/c nu/nu mice (5000 cells per mouse). Four mice were injected for each group. Mice in the control group were sacrificed upon the development of neurologic signs. All the mice bearing c-Myc knockdown glioma cells did not develop neurologic signs and were sacrificed after 100 days without evidence of tumor. Kaplan-Meier survival curves are displayed. (B) Representative photographs of hematoxylin and eosin staining of intracranial xenograft tumors (10×). (C) Xenograft tissue of the control group composed of pleomorphic cells featuring high nuclear to cytoplasmic ratios, prominent nucleoli with minimal cytoplasm, brisk mitotic activity and central geographic necrosis (asterisk, 600×). (D) The control glioma xenograft exhibited focal areas of better differentiated tumor cells with relatively more eosinophilic cytoplasm and cells with eccentric cytoplasmic profiles suggestive of a gemistocytic appearance (arrows, 400×). (E) Xenograft tumor of the control group exhibits infiltration of tumor cells into the surrounding brain tissue along the margin. The mitotically active (arrowhead) infiltrating tumor cells exhibit high nuclear to cytoplasmic ratios and elongated fibrillar cytoplasm (arrow) (600×). (F) Mouse brain injected with T3359 cells expressing c-Myc shRNA showed no evidence of tumor at the needle injection site (arrows, 200×).</p

c-Myc modulates cell cycle regulators of glioma cancer stem cells.

<p>(A) Early passage (WAF1/CIP1, cyclin D₁ (cycD1) and cyclin D₂ (cycD2) were determined by quantitative real-time PCR 3 days after infection. (B) Protein levels of c-Myc, p53, cyclin D₁, cyclin D₂, cyclin E and p21^WAF1/CIP1 were determined by immunoblotting.</p

c-Myc is highly expressed in glioma cancer stem cells.

<p>(A) CD133− and CD133+ cells were isolated from glioma surgical biopsy specimens passaged short-term in immunocompromised mice and briefly cultured. Total RNA was isolated from both CD133− and CD133+ cells. cDNA was prepared by reverse transcription. Expression of c-Myc was then determined by quantitative real-time PCR and normalized to β-actin and HPRT1. Relative mRNA levels of c-Myc in CD133− cells were assigned a value of 1. Data are represented as mean±S.E.M in this and all subsequent graphs (#: p<0.001). (B) Total cellular lysates were resolved by SDS-PAGE. Protein levels of c-Myc and Olig2 were determined by immunoblotting. Actin was blotted as the loading control. (C) Glioma cells were isolated directly from human surgical biopsy specimens, fixed in 4% paraformaldehyde following dissociation, labeled with anti-CD133-APC and anti-c-Myc-FITC, and subjected to FACS analysis. (D) Percentage of cells expressing high levels of c-Myc within either the CD133− fraction or the CD133+ fraction was demonstrated (#: p<0.001). (E) Sections of freshly frozen human glioma surgical biopsy specimens were fixed and co-stained for c-Myc (green) and Nestin (red). Nuclei were counterstained with Hoechst 33342. Representative images (630×) were demonstrated.</p

c-Myc induces cell cycle arrest of glioma cancer stem cells.

<p>(A, B) Early passage (0 phase; M2-G₀/G₁ phase; M3-S phase; M4-G2/M phase.</p

Depletion of c-Myc induces apoptosis in glioma cancer stem cells.

<p>(A, B) T3359 and (C, D) T4597 CD133− and CD133+ cells were isolated and infected as described. (A, C) Six days after infection, cells were trypsinized and quantified for apoptosis using the Annexin V-FITC Apoptosis Detection kit (Calbiochem). The percentage of FITC positive cells was determined by FACS analysis, and dead cells were excluded by propidium iodide staining. (B, D) These cells were also plated in 96-well plates at 5000 cells per well for CD133− cells and 1000 cells per well for CD133+ cells after infection and selection. Six days after infection, combined activities of caspase 3 and caspase 7 were determined by the Caspase 3/7 Luminescence Assay kit (Promega), and were normalized by the viable cell numbers determined by the CellTiter-Glo assays as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0003769#pone-0003769-g004" target="_blank">Figure 4</a>. *, p<0.05 with one-way ANOVA comparison of the control groups to the corresponding c-Myc knockdown groups.</p

Depletion of c-Myc inhibits growth of glioma cancer stem cells.

<p>(A) T3359, (B) T3832, and (C) T4597 CD133− and CD133+ cells were isolated and infected as described. Cells were then plated in 96-well plates in triplicate at 5000 cells per well for CD133− cells or 1000 cells per well for CD133+ cells. Total viable cell numbers were then determined by the CellTiter-Glo Luminescent Cell Viability Assay (Promega) daily. *, p<0.05; #, p<0.001 with one-way ANOVA comparison of the control groups to the corresponding c-Myc knockdown groups on the same day.</p

Characteristics of study participants.

<p>SBP, systolic blood pressure; DBP, diastolic blood pressure; BMI, body mass index; TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; TG, triglyceride; Glu, glucose; Cr, creatinine; ALT, alanine aminotransferase; HR, heart rate.</p><p>Values are mean±SD.</p><p>NS indicates not significant.</p

The frequencies of the β2-adrenergic receptor gene C-47T, A46G and C79G polymorphisms genotypes.

<p>*P value of the comparison of the additive genetic model using the generalized linear model.</p><p>**P value of the comparison of allelic frequencies.</p

Stratified analyses of association between the genotypes and risk of essential hypertension in the obese and the non-obese participants.

<p>ORs adjusted for gender, age, body mass index, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, serum triglyceride levels, plasma glucose level, smoking habits and drinking habits. OR, odds ratio; CI, confidence interval; SNP, single-nucleotide.</p><p>*ORs adjusted for gender was not performed in sub-group analyses of Male and Female.</p><p>P-value for the interaction between A46G genotype and body mass index on hypertension was 0.010.</p

Haplotype analyses of the β2-adrenergic receptor gene polymorphisms in hypertension and control subjects.

<p>All haplotypes with frequency greater than 1% detected in the haplotype analyses are shown in the table.</p><p>HS test, haplotype specific testing; OR, odds ratio; CI, confidence interval.</p>a<p>P values and OR values derived from comparing of a specific haplotype with the other two.</p>b<p>P values and OR values derived from comparing each haplotype with the base-line haplotype (T-A-C).</p>c<p>P value for global test comparing model with haplotypes to model without.</p