Journal: Open Biology

Article Title: The human parasite Leishmania amazonensis downregulates iNOS expression via NF-κB p50/p50 homodimer: role of the PI3K/Akt pathway

doi: 10.1098/rsob.150118

Figure Lengend Snippet: PI3K inhibition reduces p50/p50 occupancy on the iNOS promoter and increases iNOS message levels in infected macrophages. ( a ) RAW 264.7 cells were infected for 5 h with promastigotes of L. amazonensis and treated with ( a ) 10 µM LY294002 (PI3K inhibitor), ( b ) 1 µM wortmannin or ( c ) 5 µM Akti-1/2. The ChIP assay was carried out using anti-p50 antibodies. ( d ) Peritoneal macrophages were infected with L. amazonensis and/or treated with the PI3K inhibitor LY294002 and/or LPS (1 µg ml −1 ). The samples were subjected to qPCR, as previously described. * p < 0.05.

Article Snippet: To inhibit the PI3K/Akt pathway, cells were treated with 10 μM of LY294002 (Sigma-Aldrich) or 1 μM of wortmannin (Sigma-Aldrich) or 5 μM of Akt inhibitor VIII, isozyme-selective, Akti-1/2 (Santa Cruz Biotechnology) during the infection.

Techniques: Inhibition, Infection

Journal: PLoS ONE

Article Title: Stathmin Regulates Keratinocyte Proliferation and Migration during Cutaneous Regeneration

doi: 10.1371/journal.pone.0075075

Figure Lengend Snippet: (A) Real-time PCR kinetics of Stathmin mRNA expression for 8 hours after administration of HGF (20 ng/ml). For each time-point the ratio of stimulated to untreated primary human keratinocytes was calculated. Data are shown as mean +/- SEM (n=3) and were normalized to transcript levels of untreated cells. (B) Scheme depicting the two major signaling pathways activated by HGF. Chemical inhibitors Akti-1/2 (5 µM) and GW5074 (2 µM) were utilized for selective inhibition of PI3K/AKT- and Ras/Raf/MAPK-pathways. Stathmin transcript levels of keratinocytes were analyzed 1h after stimulation with HGF (20 ng/ml) and administration of both substances for 15 and 30 min, respectively. Data are shown as mean +/- SEM (n=3). (C) Quantitative western immunoblotting analysis of Stathmin and phospho-Stathmin in keratinocytes after stimulation with HGF and administration of Akti-1/2 or GW5074. Total AKT and ERK1/2 as well as phospho-AKT (pAKT) and phospho-ERK1/2 (pERK1/2) served as controls for successful HGF stimulation and pathway inhibition. Numbers indicate quantitative densitometric values of phospho-Stathmin and total Stathmin levels compared to untreated cells. (D) Influence of c-Met inhibition on total Stathmin levels and its phosphorylation status using a receptor-specific inhibitor. Primary keratinocytes were treated with the c-Met inhibitor (PHA-665752, 0.5µM). Numbers indicate quantitative densitometric values of phospho-Stathmin and total Stathmin. (E) Stathmin and c-Met protein expression in keratinocytes after siRNA-mediated inhibition of Stathmin and c-Met after 48 hours (final concentration: 20 nM). Two independent nonsense siRNAs served as negative controls. Different parts of one gel are shown (dividing lines). For all western immunoblots, actin served as loading control.

Article Snippet: Treatment with the kinase inhibitors was performed immediately before HGF administration for 15 (Akti 1/2, 5 μM, Merck, Darmstadt, Germany) or 30 minutes (GW5074, 2 μM, Sigma-Aldrich, St. Louis, USA), respectively.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Protein-Protein interactions, Inhibition, Western Blot, Phospho-proteomics, Concentration Assay, Control

Journal: Oncotarget

Article Title: Amino acid-dependent signaling via S6K1 and MYC is essential for regulation of rDNA transcription

doi: 10.18632/oncotarget.10346

Figure Lengend Snippet: A. and B. HeLa cells were amino acid and serum-starved (-AAS) for 2 hours, pre-treated with either 5 μM AKTi-1/2 or 20 nM rapamycin A. or 10 μM PF4708671 B. for 30 minutes, and then stimulated with all amino acids (AA) for 3 hours. C. HeLa cells were transfected with either non-targeting siRNA control (siCON) or pooled siRNA against S6K1 (siS6K1) for 3 days, and then starved of all amino acids and serum for 2 hours followed by amino acid stimulation for 3 hours. D. HeLa cells were treated as in B. E. HeLa cells stably expressing a doxycycline (DOX)-inducible 4E-BP1-4A mutant plasmid were treated with 1 μg/ml DOX for 24 hours, starved of all amino acids and serum for 2 hours and then stimulated with amino acids for 3 hours. A. - C. and E. In all cases cells were pulse labeled and 47/45S rRNA synthesis analyzed. Representative images and resultant graph (mean +/− SEM) of n = 3-4 experiments. Below the graph are representative immunoblotting images, n = 2-3. D. qChIP analysis to assess the Pol I loading on various regions of the rDNA. n = 3 experiments. A. * p < 0.05 compared to Lane 2; # p < 0.05, ## p < 0.01 compared to Lane 1. B. * p < 0.05 compared to Lane 5, # p < 0.05 compared to Lane 1. C. * p < 0.05 compared to Lane 2, # p < 0.05 compared to Lane 1. D. NS, not significant compared to Lane 2.

Article Snippet: Inhibitors including: 5 μM AKTi-1/2 in dimethyl sulfoxide (DMSO) (MERCK, #124017), 20 nM rapamycin in ethanol (Calbiochem, #553210), 10 μM PF4708671 in DMSO (Sigma, #PZ0143), 40 μM 6-Diazo-5-oxo-L-norleucine (DON) in water (Sigma, #D2141).

Techniques: Transfection, Stable Transfection, Expressing, Mutagenesis, Plasmid Preparation, Labeling, Western Blot