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

Journal: Molecular Cancer

Article Title: Hyperactivation of NF-κB via the MEK signaling is indispensable for the inhibitory effect of cAMP on DNA damage-induced cell death

doi: 10.1186/1476-4598-10-45

Figure Lengend Snippet: Knockdown of p65 relieves the inhibitory effect of forskolin or 8-CPT-cAMP on IR-induced cell death . Cells transfected with control siRNA or p65 siRNA were treated with or without forskolin (80 μM) or 8-CPT-cAMP (200 μM) for 30 min before exposure to IR (10 Gy). After 20 h, cells were analyzed for PI uptake by FACS (n = 4). The p values were calculated relative to cells treated with IR only: Reh, * p < .01, ** p < .04. TK6, * p < .03, ** p < .05. The histograms in the right panel depict percent inhibition of IR-induced cell death by forskolin or 8-CPT-cAMP in cells transfected with control siRNA or p65 siRNA.

Article Snippet: For siRNA transfection, Reh or TK6 cells (6 × 10 6 ) were transfected with 16 pmol Signalsilence NF-κB p65 siRNA (6261; Cell signaling Technology) or stealth RNAi for human MEK1 and MEK2 (12935-025; Invitrogen) using the nucleofection solution R and the O-17 program (Reh) or solution V and the X-05 program (TK6) with a nucleofector device (Amaxa Biosciences).

Techniques: Knockdown, Transfection, Control, Inhibition

Journal: Molecular Cancer

Article Title: Hyperactivation of NF-κB via the MEK signaling is indispensable for the inhibitory effect of cAMP on DNA damage-induced cell death

doi: 10.1186/1476-4598-10-45

Figure Lengend Snippet: cAMP enahnaces the IR-induced phosphorylation of IKKβ and nuclear translocation of p65 . (A) Reh cells were treated with or without forskolin (80 μM) for 30 min before irradiation (10 Gy). Cells were harvested at the indicated times and subjected to Western blot analysis with the indicated antibodies. The figure shows 1 representative blot of 4 experiments. (B) Reh cells were treated as in A, harvested at the indicated times and subjected to immunoblot analysis with the indicated antibodies. The figure shows 1 representative blot of 4 experiments. (C) Reh cells were treated as in A. One half of each sample was used for preparation of total cell lysate and the other half was subjected to cellular fractionation to obtain nuclear lysate. The lysates were then analyzed by immunoblotting with the indicated antibodies. The figure shows 1 representative experiment of 3. (D) Reh cells were cultured in the absence or presence of Bay 11-7082 (5 μM) for 90 min before treatment with or without forskolin (80 μM) for 30 min. Cells were then exposed to 10 Gy IR and harvested after 2 h. One half of each sample was used for preparation of total cell lysate and the other half was subjected to cellular fractionation to obtain nuclear lysate. The lysates were then analyzed by immunoblotting with the indicated antibodies. The figure shows 1 representative experiment of 3.

Article Snippet: For siRNA transfection, Reh or TK6 cells (6 × 10 6 ) were transfected with 16 pmol Signalsilence NF-κB p65 siRNA (6261; Cell signaling Technology) or stealth RNAi for human MEK1 and MEK2 (12935-025; Invitrogen) using the nucleofection solution R and the O-17 program (Reh) or solution V and the X-05 program (TK6) with a nucleofector device (Amaxa Biosciences).

Techniques: Phospho-proteomics, Translocation Assay, Irradiation, Western Blot, Cell Fractionation, Cell Culture

Journal: Molecular Cancer

Article Title: Hyperactivation of NF-κB via the MEK signaling is indispensable for the inhibitory effect of cAMP on DNA damage-induced cell death

doi: 10.1186/1476-4598-10-45

Figure Lengend Snippet: Potentiation of NF-κB DNA binding and transcriptional activity by cAMP . (A) Reh cells were treated with or without forskolin (80 μM) for 30 min before irradiation (10 Gy). Cells were harvested at the indicated times after IR and nuclear extracts were prepared. The binding activity of p65 in the nuclear extracts was then determined by the p65 TransAM assay (n = 4). * p < .01, ** p < .05 relative to cells treated with IR only. (B) Reh cells were transfected with a plasmid encoding the luciferase gene under 3 repeats of a NF-κB consensus binding site. After 24 h, cells were cultured in the presence or absence of forskolin (80 μM) for 30 min before exposure to IR (10 Gy). At the indicated times after IR, cells were harvested and the luciferase activity was measured as described in Materials and methods (n = 4). * P < .01 relative to cells treated with IR only. (C) Reh cells were transfected as in B. After 24 h, cells were cultured in the presence or absence of forskolin (80 μM), 8-CPT-cAMP (200 μM) or 8-pCPT-2'-O-Me-cAMP (400 μM) for 30 min before exposure to IR (10 Gy). Cells were harvested at 4 h after IR and luciferase activity was measured as described in Materials and methods (n = 4). * P < .01, ** P < .05 relative to cells treated with IR only. (D) Freshly isolated splenocytes from 3 × κB-luc transgenic mice were cultured in the presence or absence of forskolin (80 μM) for 30 min before exposure to IR (10 Gy). After 2 h, luciferase activity was measured as described in Materials and Methods. The boxes show the median, upper and lower quartile, and the whiskers show the range of values (n = 9, * P < .05 by Wilcoxon signed rank test when compared with cells treated with IR only). The outlier value (○) stems from one mouse.

Article Snippet: For siRNA transfection, Reh or TK6 cells (6 × 10 6 ) were transfected with 16 pmol Signalsilence NF-κB p65 siRNA (6261; Cell signaling Technology) or stealth RNAi for human MEK1 and MEK2 (12935-025; Invitrogen) using the nucleofection solution R and the O-17 program (Reh) or solution V and the X-05 program (TK6) with a nucleofector device (Amaxa Biosciences).

Techniques: Binding Assay, Activity Assay, Irradiation, Transfection, Plasmid Preparation, Luciferase, Cell Culture, Isolation, Transgenic Assay

Journal: Molecular Cancer

Article Title: Hyperactivation of NF-κB via the MEK signaling is indispensable for the inhibitory effect of cAMP on DNA damage-induced cell death

doi: 10.1186/1476-4598-10-45

Figure Lengend Snippet: Inhibition of MEK signaling alleviates the inhibitory effect of cAMP on IR-induced cell death . (A) Cells were cultured in the absence or presence of 50 μM PD 98059 alone (Reh and TK6) or together with 400 μM IBMX (EU-3) for 45 min before treatment with or without 80 μM forskolin (Reh), 200 μM 8-CPT-cAMP (TK6) or 100 μM forskolin (EU-3) for 30 min. Cells were then exposed to 10 Gy IR, harvested after 12 h (EU-3) or 20 h (Reh and TK6) and analyzed for PI uptake by FACS (n = 4). The p values were calculated relative to cells treated with IR only: Reh, * p < .02. TK6, * p < .01, ** p < .03. EU-3, * p < .01, ** p < .04. The histograms in the right panel depict percent inhibition of IR-induced cell death by forskolin, 8-CPT-cAMP or forskolin and IBMX in the presence or absence of PD 98059. (B) Reh cells were transfected with control siRNA or siRNAs against MEK1 and MEK2. Cells were then treated with or without forskolin (80 μM) for 30 min before exposure to IR (10 Gy). After 20 h, cells were analyzed for PI uptake by FACS (n = 4). * p < .01, ** P < .03 relative to cells treated with IR only. The histogram in the right panel depicts percent inhibition of IR-induced cell death by forskolin in cells transfected with control siRNA or MEK1 and MEK2 siRNAs.

Article Snippet: For siRNA transfection, Reh or TK6 cells (6 × 10 6 ) were transfected with 16 pmol Signalsilence NF-κB p65 siRNA (6261; Cell signaling Technology) or stealth RNAi for human MEK1 and MEK2 (12935-025; Invitrogen) using the nucleofection solution R and the O-17 program (Reh) or solution V and the X-05 program (TK6) with a nucleofector device (Amaxa Biosciences).

Techniques: Inhibition, Cell Culture, Transfection, Control