Journal: PLoS ONE

Article Title: Overexpression of miR-125a in Myelodysplastic Syndrome CD34 + Cells Modulates NF-κB Activation and Enhances Erythroid Differentiation Arrest

doi: 10.1371/journal.pone.0093404

Figure Lengend Snippet: NF-κB activation was determined after 48 hours from transfection of Meg-01 cells with miRNA mimics and luciferase vectors. Results are relative to cells transfected with mock RNA and expressed as mean ± SEM of n = 3 independent experiments. Statistical significance: *P<0.05; ***P<0.001.

Article Snippet: Cell Line Nucleofector Solution R (Lonza, Basel, Switzerland) was used to nucleofect KG1 cells with pGL4.32[luc2P/NF-κB-RE/Hygro] and pRL-TK vectors (Promega, Madison, WI) using program V-001 of Amaxa Nucleofector and the corresponding reagent kit (Lonza, Basel, Switzerland).

Techniques: Activation Assay, Transfection, Luciferase

Journal: PLoS ONE

Article Title: Overexpression of miR-125a in Myelodysplastic Syndrome CD34 + Cells Modulates NF-κB Activation and Enhances Erythroid Differentiation Arrest

doi: 10.1371/journal.pone.0093404

Figure Lengend Snippet: NF-κB activation was measured after 24 hours from nucleofection of KG1 cells with the luciferase vectors and treatment with ASO. Results are expressed as relative to cells transfected with mock and represent mean ± SEM of n = 4 independent experiments. Method disclosure: technical problems regarding the endogenous Renilla control were experienced during these luciferase assays; only one experiment out of four efficiently expressed the Renilla luciferase and could be properly normalized. Because normalized results were almost identical to non-normalized data, we conducted a joint statistical analysis of the four experiments. Statistical significance: ***P<0.001.

Article Snippet: Cell Line Nucleofector Solution R (Lonza, Basel, Switzerland) was used to nucleofect KG1 cells with pGL4.32[luc2P/NF-κB-RE/Hygro] and pRL-TK vectors (Promega, Madison, WI) using program V-001 of Amaxa Nucleofector and the corresponding reagent kit (Lonza, Basel, Switzerland).

Techniques: Activation Assay, Luciferase, Transfection

Journal: PLoS ONE

Article Title: Overexpression of miR-125a in Myelodysplastic Syndrome CD34 + Cells Modulates NF-κB Activation and Enhances Erythroid Differentiation Arrest

doi: 10.1371/journal.pone.0093404

Figure Lengend Snippet: Discontinuous arrows represent speculations and unknown mechanisms; consecutive arrows represent known pathways that do not need to be explained for the understanding of the figure. ( A ) In the absence of TLR signaling, the miR-99b/let-7e/miR-125a cluster (and very likely also miR-125b) is coordinately upregulated, via transcriptional activation by either NF-κB as a part of a positive feedback loop or by other transcription factors (TF). miR-125a, presumably in collaboration with miR-99b, enhances NF-κB activation, probably through the inhibition of the TNF-induced NF-κB inhibitor TNFAIP3 and/or other inhibitors, such as IκBε. Thus, the expression of the miR-99b/let-7e/miR-125a cluster may favor survival of hematopoietic cells, protecting them from the deadly effects of TNF-α. ( B ) Upon TLR signaling, NF-κB is activated through a cascade of adaptor proteins. Under these conditions, the miR-99b/let-7e/miR-125a cluster is not expressed (or expressed at low levels) but the expression of miR-125a might be independently induced by unknown (maybe disease-related) mechanisms, such as the initiation of transcription at alternative promoter regions or, more likely, the differential processing of the primary transcript in one or more stages of miRNA biogenesis. Highly increased levels of miR-125a could preferentially target the mRNA of one or more genes downstream of TLRs and participate in the negative modulation of proinflammatory signaling. In MDS, elevated miR-125a levels in cells with normal TLR signaling (A) would favor sustained NF-κB activation and pro-survival effects; while in those cells with increased TLR/MyD88 levels and activation (B), high miR-125a levels would negatively modulate NF-κB activation. However, in this case, the hyperactivation of the TLR/MyD88/NF-κB pathway would probably mask the inhibitory effects of miR-125a. Additionally, in both cases, (A) and (B), overexpression of miR-125a in MDS patients would favor aberrant differentiation so its effects in either case would be detrimental for the course of the disease.

Article Snippet: Cell Line Nucleofector Solution R (Lonza, Basel, Switzerland) was used to nucleofect KG1 cells with pGL4.32[luc2P/NF-κB-RE/Hygro] and pRL-TK vectors (Promega, Madison, WI) using program V-001 of Amaxa Nucleofector and the corresponding reagent kit (Lonza, Basel, Switzerland).

Techniques: Activation Assay, Inhibition, Expressing, Over Expression

Journal: Blood

Article Title: Activation of cAMP signaling inhibits DNA damage-induced apoptosis in BCP-ALL cells through abrogation of p53 accumulation.

doi: 10.1182/blood-2009-02-204883

Figure Lengend Snippet: Figure 6. p53 is required for the inhibitory effect of cAMP on IR-induced apoptosis. (A) Reh cells were transfected with the pXJ-E6 plasmid expressing the HPV16-strain E6-protein, empty vector, siRNA-p53, or control siRNA-A. Cells were then exposed to 10 Gy IR or left untreated. After 12 hours, a portion of the cells was stained with PI for cell death analysis, and the remaining portion was subjected to immunoblotting with the indicated antibodies (n 3). *P .05; **P .01. (B) Reh cells were incubated with or without forskolin for 30 minutes before addition of menadione or staurosporine, and examined for PI uptake after 18 hours. Because menadione interferes with PI fluorescence, the shift in scatter profile was used to discriminate between dead and viable menadione-treated cells (menadione, n 3; staurosporine, n 4).

Article Snippet: Reh cells (6 106) were transfected with 10 g pXJ-E6 plasmid encoding the HPV16 E6 protein or with 16 pmol p53 or control siRNA (sc-29435 or sc-37007, respectively; Santa Cruz Biotechnology) using the Nucleofection solution R and the G-16 program with a Nucleofector device (Amaxa Biosciences).

Techniques: Transfection, Plasmid Preparation, Expressing, Control, Staining, Western Blot, Incubation