Journal: PLoS ONE

Article Title: NF-κB-Induced IL-6 Ensures STAT3 Activation and Tumor Aggressiveness in Glioblastoma

doi: 10.1371/journal.pone.0078728

Figure Lengend Snippet: A & B, Xenograft X1046 cells were disaggregated into single cells and briefly propagated as neurospheres in vitro . Cells were pre-treated with AZD1480 (1 µM) and/or WA (5 µM) for 2 h prior to TNF-α (10 ng/ml) for 0.25 or 2 h (A) or 2 h (B). Cells were lysed and immunoblotted with the indicated Ab (A), or RNA was isolated followed by generation of cDNA and qRT-PCR was performed for IL-6 (B). Densitometric values of p-p65, p-IKKα/β and p-STAT3 were normalized to total p65, total IKKα/β and total STAT3, respectively. Data are shown as replicates of three. *, p<0.05. C, Xenograft X1066 cells were treated with the indicated doses of AZD1480 and/or WA for 48 h, and the WST-1 cell viability assay was performed. Data are shown as replicates of three. *p<0.05. D, Nude mice were injected intracranially with Xenograft X1016 cells. Starting at day 3, mice were treated with vehicle (n = 5), AZD1480 (30 mg/kg, twice a day, n = 5), WA (4 mg/kg, alternate days, n = 5) or both AZD+WA (n = 5) for three weeks. Survival was measured, and mice were euthanized at moribund. *, p<0.05 (LogRank).

Article Snippet: Antibodies against phospho-p65 Ser536 (p-p65 (S536)), phospho-STAT3 Tyr705 (p-STAT3 (Y705)), STAT3, phospho-IKKα/β (S176/180) and IKKα/β were purchased from Cell Signaling Technology (1∶1,000; Beverly, MA).

Techniques: In Vitro, Isolation, Quantitative RT-PCR, Viability Assay, Injection

Journal: PLoS ONE

Article Title: Induction of Erythroid Differentiation in Human Erythroleukemia Cells by Depletion of Malic Enzyme 2

doi: 10.1371/journal.pone.0012520

Figure Lengend Snippet: K562 cells with or without ME2 knockdown were lysed with RIPA lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 1 mM EGTA) containing 1 mM PMSF and a protease inhibitor cocktail and subjected to centrifugation at 15,000× g for 10 min at 4°C to remove debris. After lysis, equal aliquots of protein lysate were resolved by Western blotting. Western blots were probed with anti-phospho-ERK1/2, anti-ERK1, anti-p-AKT308, anti-AKT472, anti-AKT1/2, anti-GATA-1, anti-vimentin and anti-β-tubulin. A, phospho-ERK1/2 activity in ME2 knockdown K562 cells. B, Phospho-AKT detection in ME2 knockdown K562 cells. C, 10 µM PI3K inhibitor, LY294002, inhibits p-AKT activity. D, LY294002 rescue of differentiation in ME2 knockdown K562 cells. E, The effect of LY294002 on the proliferation of K562 cells with or without ME2 knockdown. F, The expression difference of GATA-1 and vimentin in ME2 knockdown cells. Data are representative of three independent experiments.

Article Snippet: The anti-GATA-1, phospho-ERK1/2, phospho-AKT308, phospho-AKT473 and AKT1/2 polyclonal antibodies were purchased from Cell Signaling Technology.

Techniques: Lysis, Protease Inhibitor, Centrifugation, Western Blot, Activity Assay, Expressing

Journal: BMC Neuroscience

Article Title: Anaesthesia generates neuronal insulin resistance by inducing hypothermia

doi: 10.1186/1471-2202-9-100

Figure Lengend Snippet: Insulin sensitivity is maintained during anaesthesia if hypothermia is prevented . Mice were injected i.p. with ketamine/Xylazine (K/X), and body temperature maintained at 37°C. Half of the group (n = 4) were given insulin by icv injection, the other half (n = 4) received saline 30 mins prior to sacrifice and rapid removal and dissection of the brain. Equal amounts of cortical protein were subjected to western blot using the antibodies shown (A), and results from each treatment group quantified by densitometry (B). The mean ± SD, relative to the control animals, is shown. Significant differences between control and each treatment group are given, *p < 0.05, **p < 0.005. Phosphorylation of ERK and PKB was also compared between animals sacrificed without anaesthesia and those with anaesthesia but whose body temperature was maintained at 37°C (C).

Article Snippet: Antibodies to phospho-PKB (Thr308), phospho-PKB (Ser473), phospho-ERK (Thr202/Tyr204), were from Cell Signaling Technology (Beverly, MA, U.S.A.), antibodies to total PKB, ERK1/2 and GSK3α/β were from Upstate Biotechnologies (Lake Placid, NY, U.S.A.).

Techniques: Injection, Dissection, Western Blot