Immunoblotting revealed dose- and time-dependent increases in Bec

Immunoblotting revealed dose- and time-dependent increases in Beclin 1 expression in cells exposed to DHA (Figure  3B). These findings demonstrated that treatment with DHA activates JNK and Beclin 1 in both pancreatic cancer cell lines in a dose- and time-dependent manner. Up-regulation of JNK expression following DHA treatment depends on ROS JNK pathway over-activation is crucial to many processes leading to cell death, including chronic and acute oxidative stress. Although

ROS can increase JNK signaling via the activation of upstream kinases or the inactivation of phosphatases, other unknown mechanisms find more are likely to contribute to ROS-induced JNK increases in pancreatic cancer cells. To exclude the possibility that other mechanisms were responsible for our observations, we measured ROS levels in response to DHA. ROS were increased after DHA treatment and did not differ between the two tested cell lines (Figure  4A). Figure 4 JNK expression induced by DHA is dependent on ROS generation. (A) BxPC-3 and PANC-1 cells were treated with 50 μmol/L DHA for different times, and then subjected to flow cytometry to measure ROS levels, as described in the Materials and Methods section. (B, C) BxPC-3 and PANC-1 cells were treated with 50 μmol/L DHA for 24 h in the presence or absence of 10 μmol/L

SP600125 or 10 mmol/L NAC pretreatment for 1 h and then subjected to flow cytometry to Cilomilast measure the levels of ROS. (D) immunoblot analysis of the phospho-JNK levels in BxPC-3 and PANC-1 cells treated with the indicated concentrations

of DHA for 24 h in the presence or absence of 10 mmol/L NAC. *P < 0.05. To further determine whether DHA treatment requires JNK activation to generate ROS, we pre-treated BxPC-3 cells with SP600125 (a specific JNK inhibitor) for 1 h, before exposing them to DHA. In contrast to DHA treatment alone, SP600125 pretreatment prevented alterations in ROS levels (Figure  4B). To examine whether ROS inhibition impacted JNK signaling, we compared JNK activation with or without N-acetyl-L-cysteine (NAC, a ROS inhibitor). NAC pretreatment significantly lowered intracellular ROS compared with DHA-treated from cells (Figure  4C). More importantly, the degree of JNK activation after DHA treatment was decreased in the cells pretreated with NAC (Figure  4D), and this decreased JNK activation was related to the inhibition of ROS formation. These results indicate that JNK expression following DHA treatment depends on ROS. Inhibition of JNK expression down-regulates beclin 1 and reduces autophagy To further assess the role of JNK in DHA-induced autophagy, cells were pretreated with SP600125 (10 mM) for 1 h, and were then exposed to DHA. In contrast to DHA alone, SP600125 pretreatment blocked the increase in LC3-II induced by DHA (Figure  5A). Furthermore, SP600125 treatment decreased the punctate foci of LC3 in the cytoplasm (Figure  5B).

Colonies were grown for 3 days at 37°C Hydrated lasR mutant biof

Colonies were grown for 3 days at 37°C. Hydrated lasR mutant biofilms do not show altered architecture The involvement of pel in the wrinkled colony morphology of the ZK lasR mutant suggested that it might exhibit generally altered

biofilm architecture. We investigated pellicle formation of standing cultures as well as biofilm formation in microtiter plates and flow-cells. Flow-cell biofilms of the wild-type and the lasR mutant after 3 days of growth are shown in Figure 5. Neither assay revealed any differences between the two strains. This is consistent with recent results by Colvin et al., who also found no defect in attachment or biofilm development for a pel mutant of strain PAO1 [56]. There is a difference in the degree of buy EPZ-6438 hydration in the three biofilm assays we employed. Submerged flow-cell biofilms are fully saturated and hydrated, pellicles and microtiter plate biofilms that form at the air-liquid interface are somewhat

less hydrated, whereas colonies on agar Tamoxifen price are the least hydrated [57]. It is possible that the observed phenotype only manifests under conditions of low hydration. Figure 5 Flow-cell biofilms. CLSM images of flow-cell grown biofilms of the ZK wild-type (WT) and the lasR mutant at 37°C after 3 days. The large panel shows the horizontal cross-section and the small panel shows the vertical cross-section of the biofilm. The lines in the panels indicate the planes of the cross-sections. Suppressor mutagenesis implicates the pqs pathway Transposon mutagenesis was performed in the ZK lasR mutant background to identify the regulatory link between the las QS system and colony morphology. Around 10,000 mutants were screened for reversion to a smooth phenotype. We identified 38 mutants, and mapped very transposon insertions in 25 (Additional file 2: Table S2). We found 9 transposon insertions in the pqsA-D genes of the AQ biosynthesis operon and one insertion in the gene encoding the transcriptional regulator PqsR that activates pqsA-E expression (Figure 6). Given the large fraction of hits (10 out of 25 or 40%), the role of the pqs operon was apparent even without mapping

the remaining transposon mutants. We did not identify any insertions in pqsH, which promotes the conversion of Series A (4-hydroxyalkyl quinolines) to Series B (3,4 dihydroxyalkyl quinolines) congeners nor in pqsE, which encodes a putative global regulator [20, 58]. Surprisingly, we also did not identify a transposon insertion in the pel operon, although our data in Figure 3 show that the lasR pel mutant forms a smooth colony. We found that this mutant displayed very slight wrinkling under the conditions employed for the high throughput screen, in which our primary focus was on the identification of the most obvious smooth revertants. Figure 6 The pqs locus and transposon insertions in associated suppressor mutants. Horizontal arrows represent the genes of the pqsA-E operon, the pqsR transcriptional regulatory gene, and the pqsH gene.