Frank M. J. Jacobs (Rudolf Magnus Institute of Neuroscience, University Medical Center, Utrecht, the Netherland) and was reported previously [18]. Berberine was purchased from Chengdu Must Biotechnology (Chengdu,

China). Cell culture The NSCLC cell lines (A549, PC9, H1650 and H1299) were obtained from the Chinese Academy of Sciences Cell Torin 1 clinical trial Bank of Type Culture Collection (Beijing, China) and the Cell Line Bank at the Laboratory Animal Center of Sun Yat-sen University starting March 2012 (Guangzhou, China) and grown in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, HEPES buffer, 50 IU/mL penicillin/streptomycin, and 1 μg amphotericin (complete medium). All cell lines have been tested and authenticated for absence of Mycoplasma, genotypes, drug response, and morphology using a commercial available kit www.selleckchem.com/products/mek162.html (Invitrogen, Shanghai, China) in the Laboratory and in the Animal Center at Sun Yat-sen University. The BBR was dissolved in a small amount of dimethylsulfoxide [DMSO, maximum concentration, 0.1% (v/v)], which was then added to complete cell culture medium prior to addition to sub confluent cells. Cells treated with vehicle only (DMSO, 0.1% in media) was served as control. Cell viability assay NSCLC cells were seeded in 96-well plates at 5 × 103 cells/well, and incubated at 37°C in complete medium for 24 h before the treatment. NSCLC cells were treated with SB203580, PD98059, or pifithrin-α

for 2 h or were transfected with control, or p53 and FOXO3a siRNAs for 24 h before exposure of the cells to BBR for an additional 24 h. Afterwards, cell viability were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay according to the instruction from the provider. Cell cycle analysis NSCLC cells were cultured in 6-well plates at 2 × 105 cells/well and treated with increased doses of BBR for 24 h or with SB203580, PD98059, or pifithrin-α for 2 h, followed by BBR for an additional 24 h. Afterwards, the cells were harvested, washed twice with phosphate-buffered

saline (PBS), and resuspended in 500 μL of cold PBS and ethanol (1.5 mL) for 2 h at 4°C. The fixed cells were incubated O-methylated flavonoid in 1 mL of 0.1% sodium citrate containing propidium iodide (PI) 0.05 mg and 50 μg RNase for 30 min at room temperature (RT) in the dark. The cell cycle analysis was detected by flow cytometry (FC500, Beckman Coulter, FL, USA), and the proportion (percentage) of cells within the G1, S, and G2/M phases of the cell cycle were analyzed using the MultiCycle AV DNA Analysis software (Phoenix Flow Systems). Western blot analysis NSCLC cells were harvested, washed and lysed with 1 × RAPI buffer. Protein concentrations were determined by the Thermo BCA protein assay Kit. Equal amounts of protein from cell lysates were separated on 10% and 12% SDS polyacrylamide gels, and transferred onto polyvinylidene fluoride membranes.

Since P. stutzeri A1501 was originally isolated from paddy soil and because it contains sets of genes for the β-ketoadipate pathway, it should be able to

utilize aromatic compounds. In our study, we observed that this strain can aerobically degrade benzoate and 4-hydroxybenzoate. As the complete genome of P. stutzeri A1501 was sequenced recently [20], we mapped the genes encoding the peripheral pathways for the catabolism of 4-hydroxybenzoate (pob) and benzoate (ben) in the A1501 chromosome (Figure 1A). In many soil bacteria, these peripheral pathway enzymes channel the individual substrates into one of the two branches of the β-ketoadipate CYT387 concentration pathway, namely the catechol and protocatechuate branches. Sequence comparison indicated that A1501 has genes encoding all of the enzymes involved in the two branches of the β-ketoadipate pathway. The catechol (cat genes) and the protocatechuate branches (pca genes) converge at β-ketoadipate enol-lactone. One set of enzymes, which are encoded by

pcaDIJF, completes the conversion of β-ketoadipate enol-lactone to tricarboxylic acid WZB117 mouse cycle intermediates (Figure 1B). Figure 1 The catechol and protocatechuate branches of the β-ketoadipate pathway and its regulation in P. stutzeri A1501. (A) Localization of the gene clusters involved in degradation of benzoate and 4-hydroxybenzoate on a linear map of the chromosome. (B) Predicted biochemical steps for the catechol and protocatechuate pathways in P. stutzeri A1501. The question mark indicates an unknown mechanism that may be involved in the regulation of cat genes. Inactivation of pcaD is shown by “”× “” and accumulations of the intermediates catechol and cis, cis-muconate in the supernatants of the

pcaD mutant are shown by red vertical arrows. Genes whose expression is under catabolite repression control (Crc) are indicated by “”⊥”". In the A1501 genome, the cat genes are chromosomally Erastin in vitro linked with the ben genes and form an 11.5 kb supercluster (PST1666-PST1676). The deduced amino acid sequence of BenR in A1501 shows high similarity (61% identity) to the P. fluorescens Pf-5 BenR protein. However, the catR gene, which positively regulates the catBC and catA operons in other strains [12, 25], is absent in A1501 (Figure 2A). Additionally, the pca genes in P. stutzeri A1501 are contiguous, whereas the pca genes are scattered over several portions of the genome in other Pseudomonas species, such as P. entomophila [21], P. aeruginosa [26], P. fluorescens [27]and P. putida [2] (Figure 2B). PcaR is an Icl family protein and has been reported to regulate most of the pca genes in the protocatechuate branch of the β-ketoadipate pathway in P. putida [12, 28, 29]. In contrast to other Pseudomonas strains, pcaR is located immediately upstream of pcaI in A1501 (Figure 2B). The deduced amino acid sequence of A1501 PcaR shows 85% identity to that of P. putida KT2440.

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