Development of a Multipurpose GATEWAY-Based Lentiviral Tetracycline-Regulated Conditional RNAi System (GLTR)

Sigl, Reinhard; Ploner, Christian; Shivalingaiah, Giridhar; Kofler, Reinhard; Geley, Stephan

doi:10.1371/journal.pone.0097764

Cited by 28 publications

(38 citation statements)

References 33 publications

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“…After 48 and 72 h viral supernatants were harvested, sterile filtered and supplemented with 4 µg/ml polybrene. Five × 10 5 target cells (RAW264.7) were infected with 0.5 ml viral supernatant for 6 h. The newly designated RAW.sh cells were transfected with the lentiviral expression vector pGLTR‐X‐S together with the packaging vectors and Metafectene™ (Sigl et al, ). This allows dual selection for both GFP (via FACS sorting) and puromycin (Puro), before their knockdown efficiency was determined, and the cells were then used for subsequent experiments.…”

Section: Methodsmentioning

confidence: 99%

Heme oxygenase 1 controls early innate immune response of macrophages toSalmonellaTyphimurium infection

Mitterstiller

Haschka

Dichtl

et al. 2016

Cellular Microbiology

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Macrophages are central for the immune control of intracellular microbes. Heme oxygenase 1 (HO-1, hmox) is the first and rate limiting enzyme in the breakdown of heme originating from degraded senescent erythrocytes and heme-proteins, yielding equal amounts of iron, carbon monoxide and biliverdin. HO-1 is strongly up-regulated in macrophages in response to inflammatory signals, including bacterial endotoxin. In view of the essential role of iron for the growth and proliferation of intracellular bacteria along with known effects of the metal on innate immune function, we examined whether HO-1 plays a role in the control of infection with the intracellular bacterium Salmonella Typhimurium. We studied the course of infection in stably-transfected murine macrophages (RAW264.7) bearing a tetracycline-inducible plasmid producing hmox shRNA and in primary HO-1 knockout macrophages. While uptake of bacteria into macrophages was not affected, a significantly reduced survival of intracellular Salmonella was observed upon hmox knockdown or pharmacological hmox inhibition, which was independent of Nramp1 functionality. This could be traced to limitation of iron availability for intramacrophage bacteria along with enhanced stimulation of innate immune effector pathways, including the formation of reactive oxygen and nitrogen species and increased TNF-α expression. Mechanistically, these latter effects result from intracellular iron limitation with subsequent activation of NF-κB and further inos, tnfa and p47phox transcription along with reduced formation of the anti-inflammatory and radical scavenging molecules, CO and biliverdin as a consequence of HO-1 silencing. Taken together our data provide novel evidence that the infection-driven induction of HO-1 exerts detrimental effects in the early control of Salmonella infection, whereas hmox inhibition can favourably modulate anti-bacterial immune effector pathways of macrophages and promote bacterial elimination.

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Section: Methodsmentioning

confidence: 99%

Heme oxygenase 1 controls early innate immune response of macrophages toSalmonellaTyphimurium infection

Mitterstiller

Haschka

Dichtl

et al. 2016

Cellular Microbiology

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“…Three individual shRNAs against AGMO (shAGMO506, shAGMO847, shAGMO1699) and one shRNA against GCH1 (shGCH1) were used to generate RAW264.7 knockdown cell lines according to ref. 33. A control line expressing shRNA against luciferase (shLUC) was generated similarly.…”

Section: Methodsmentioning

confidence: 99%

Tetrahydrobiopterin and alkylglycerol monooxygenase substantially alter the murine macrophage lipidome

Watschinger

Keller

McNeill

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Tetrahydrobiopterin is a cofactor synthesized from GTP with well-known roles in enzymatic nitric oxide synthesis and aromatic amino acid hydroxylation. It is used to treat mild forms of phenylketonuria. Less is known about the role of tetrahydrobiopterin in lipid metabolism, although it is essential for irreversible ether lipid cleavage by alkylglycerol monooxygenase. Here we found intracellular alkylglycerol monooxygenase activity to be an important regulator of alkylglycerol metabolism in intact murine RAW264.7 macrophage-like cells. Alkylglycerol monooxygenase was expressed and active also in primary mouse bone marrowderived monocytes and "alternatively activated" M2 macrophages obtained by interleukin 4 treatment, but almost missing in M1 macrophages obtained by IFN-γ and lipopolysaccharide treatment. The cellular lipidome of RAW264.7 was markedly changed in a parallel way by modulation of alkylglycerol monooxygenase expression and of tetrahydrobiopterin biosynthesis affecting not only various ether lipid species upstream of alkylglycerol monooxygenase but also other more complex lipids including glycosylated ceramides and cardiolipins, which have no direct connection to ether lipid pathways. Alkylglycerol monooxygenase activity manipulation modulated the IFN-γ/lipopolysaccharide-induced expression of inducible nitric oxide synthase, interleukin-1β, and interleukin 1 receptor antagonist but not transforming growth factor β1, suggesting that alkylglycerol monooxygenase activity affects IFN-γ/lipopolysaccharide signaling. Our results demonstrate a central role of tetrahydrobiopterin and alkylglycerol monooxygenase in ether lipid metabolism of murine macrophages and reveal that alteration of alkylglycerol monooxygenase activity has a profound impact on the lipidome also beyond the class of ether lipids. alkylglycerols | lipidomics | macrophages | RAW264.7 | tetrahydrobiopterin

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“…The lentiviral plasmid for conditional MCL1 silencing was generated by GATEWAY-based recombination of sequence verified pENTR-THT-MCL1 [45] (targeting sequence 5′-CCATTAGCAGAAAGTATCA-3′ cloned into pENTR-THT-I) with pGLTR-X [46]. The generation of lentiviral plasmid for conditional MCL1 (pHR-tetCMV-hMCL-ires-GFP) was described elsewhere [45].…”

Section: Methodsmentioning

confidence: 99%

Mechanistic rationale for MCL1 inhibition during androgen deprivation therapy

et al. 2015

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Androgen deprivation therapy induces apoptosis or cell cycle arrest in prostate cancer (PCa) cells. Here we set out to analyze whether MCL1, a known mediator of chemotherapy resistance regulates the cellular response to androgen withdrawal. Analysis of MCL1 protein and mRNA expression in PCa tissue and primary cell culture specimens of luminal and basal origin, respectively, reveals higher expression in cancerous tissue compared to benign origin. Using PCa cellular models in vitro and in vivo we show that MCL1 expression is upregulated in androgen-deprived PCa cells. Regulation of MCL1 through the AR signaling axis is indirectly mediated via a cell cycle-dependent mechanism. Using constructs downregulating or overexpressing MCL1 we demonstrate that expression of MCL1 prevents induction of apoptosis when PCa cells are grown under steroid-deprived conditions. The BH3-mimetic Obatoclax induces apoptosis and decreases MCL1 expression in androgen-sensitive PCa cells, while castration-resistant PCa cells are less sensitive and react with an upregulation of MCL1 expression. Synergistic effects of Obatoclax with androgen receptor inactivation can be observed. Moreover, clonogenicity of primary basal PCa cells is efficiently inhibited by Obatoclax. Altogether, our results suggest that MCL1 is a key molecule deciding over the fate of PCa cells upon inactivation of androgen receptor signaling.

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Development of a Multipurpose GATEWAY-Based Lentiviral Tetracycline-Regulated Conditional RNAi System (GLTR)

Cited by 28 publications

References 33 publications

Heme oxygenase 1 controls early innate immune response of macrophages toSalmonellaTyphimurium infection

Heme oxygenase 1 controls early innate immune response of macrophages toSalmonellaTyphimurium infection

Tetrahydrobiopterin and alkylglycerol monooxygenase substantially alter the murine macrophage lipidome

Mechanistic rationale for MCL1 inhibition during androgen deprivation therapy

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