Impact of Sphingosine Kinase-1 knock out in neonatal mice lungs
ABSTRACT: Sphingosine Kinase-1 knock out protects against hyperoxic lung injury One day old Wild type (WT) control and Sphingosine Kinase-1 knock out (SphK-1 KO)pups exposed to room air (RA) or hyperoxia (HO). Microarray based profiling of lung tissue after 7 days of hyperoxia of 75% Overall design: WT RA, WT HO, SphK-1 KO RA, and Sph K-1 KO HO studied in triplicates
Project description:Our previous data obtained by using immunohistochemistry showed, that Fgf10+/- (50% Fgf10 expression compared to WT) in hyperoxic condition at postnatal day 3 (P3) compared to WT has less vessel count in the lung and less muscularization of small capillaries in the lung. Furthermore, Fgf10+/- showed a drastic increase in mortality upon hyperoxic lung injury. Main question to be answer by this experiment is as followed: Does Fgf10+/- mice after hyperoxia from P0-P3 show different expression profiles at P3 compared to WT? To adress this question we harvest lungs at P3 from WT and Fgf10+/- after hyperoxia treatment from P0-P3. For this the mice were sacrificed by Ketamin/ Dormitor ip, lungs were perfused transcardiac with PBS and directly frozen in liquid nitrogen. Overall design: Two-condition experiment (genotype x treatment): wild-type mice and Fgf10+/- mice are kept in either normoxic or hyperoxic conditions. Sample size: 4 per group (16 total)
Project description:Sphingosine 1-phosphate (S1P) is a bioactive lipid whose levels are tightly regulated by its synthesis and degradation. Intracellularly, S1P is dephosphoryled by the actions of two S1P-specific phosphatases, sphingosine 1-phosphate phosphatase 1 and 2. To identify the physiologic functions of S1P phosphatase 1, we have studied mice with its gene, Sgpp1, deleted. Sgpp1-/- mice appeared normal at birth but during the first week of life, they exhibited stunted growth, suffered desquamation, and most died before weaning. Interestingly, the epidermal permeability barrier developed normally during embryogenesis. Sgpp1 -/- pups and surviving adults exhibited epidermal hyperplasia and abnormal expression of keratinocyte differentiation markers. Keratinocytes isolated from Sgpp1 -/- skin had increased intracellular S1P levels, and expressed a gene expression profile that indicated enhanced differentiation. The results reveal S1P metabolism as a regulator of keratinocyte differentiation and epidermal homeostasis. Comparison of KO vs. WT with five replications per treatment sample
Project description:To detect sex-specific differences in gene expression in a model of hyperoxic lung injury in adult C56BL/6J mice. In this dataset, we include the expression data obtained from lungs from 8-10 week old male and female WT C57BL/6J mice exposed to hyperoxia for 48 hours amd room air controls.These data were used to determine differences in transcriptome between male and female mice after hyperoxia exposure 12 total samples were analyzed. We generated the following pairwise comparisons: 5 comparisons (M/F: male/female, A/O: air/oxygen): 1) M_O - M_A, 2) F_O - F_A, 3) M_O - F_O, 4) M_A - F_A, 5) (M_O - M_A) - (F_O - F_A). Genes with an FDR≤5% and a fold-change ≥1.4 were selected.
Project description:Mastocytosis is a disorder resulting from an abnormal mast cell (MC) accumulation in tissues that is often associated with the D816V mutation in KIT, the tyrosine kinase receptor for stem cell factor. Therapies available to treat aggressive presentations of mastocytosis are limited, thus exploration of novel pharmacological targets that reduce MC burden is desirable. Since increased generation of the lipid mediator sphingosine-1-phosphate (S1P) by sphingosine kinase (SPHK) has been linked to oncogenesis, we studied the involvement of the two SPHK isoforms (SPHK1 and SPHK2) in the regulation of neoplastic human MC growth. While SPHK2 inhibition prevented entry into the cell cycle in normal and neoplastic human MCs with minimal effect on cell survival, SPHK1 inhibition caused cell cycle arrest in G2/M and apoptosis, particularly in D816V-KIT MCs. This was mediated via activation of the DNA damage response cascade, including phosphorylation of the checkpoint kinase 2 (CHK2), CHK2-mediated CDC25c depletion and p53 activation. Combination treatment of SPHK inhibitors with KIT inhibitors showed greater growth inhibition of D816V-KIT MCs than either inhibitor alone. Furthermore, inhibition of SPHK reduced the number of malignant bone marrow MCs from patients with mastocytosis and the growth of D816V-KIT MCs in a xenograft mouse model. Our results reveal a role for SPHK isoforms in the regulation of growth and survival in normal and neoplastic MCs and suggest a regulatory function for SPHK1 in the DNA damage response in MCs with KIT mutations. The findings also suggest that targeting the SPHK/S1P axis may provide an alternative to tyrosine kinase inhibitors, alone or in combination, for treatment of aggressive mastocytosis and other hematological malignancies associated with the D816V-KIT mutation. Overall design: HMC-1.2 cells cultured with vehicle, SPHK1 inhibitor (SPHK1-I) or SPHK2 inhibitor (SPHK2-I) for 24 h. Each treatment was done in duplicate for a tottal number of 6 samples
Project description:total RNA from mouse (male c57BL/6) spleen labeled with Cy3 vs total RNA from mouse (male c57BL/6) B cells treated with Sphingosine-1-phosphate labeled with Cy5- time course with repeats Keywords: ordered
Project description:We compared the differentially expressed genes between the F9 Wt cells and F9 RAR gamma knock out cells before and after RA treatment. 3 replicates for each conditions. We also identified the RA responsive genes in the F9 Wt cells. Experiment Overall Design: F9 wt cells and F9 RAR gamma knock out cells are treated with RA (1 uM) or ethanol for 24 hours. 3 replicates for each condition.
Project description:Neuroblastoma is an embryonal neoplasm that remains of dramatic prognosis in its aggressive forms. Activating mutations of the ALK tyrosine kinase receptor have been identified in sporadic and familial cases of this cancer. We generated knock-in mice carrying an F1178L Alk mutation corresponding to the ALK F1174L mutation observed in neuroblastoma patients We used microarrays to detail the global programme of gene expression underlying the impact of ALK F1178L mutation on sympathetic nervous system ganglia We selected sympathetic nervous system ganglia (superior cervical and stellate ganglia) for RNA extraction and hybridization on Affymetrix microarrays. We profiled a total number of 12 Po (4 Wt; 4 Hz; 4 Ho) and 5 P18 (3 Wt; 2 Hz)
Project description:KSHV is a principal causative agent of Kaposi's Sarcoma (KS). Despite this knowledge about the close relationship between sphingolipid metablism and solid tumors development, the role of sphingolipid metablism in KSHV-related malignancies remains mostly unclear. We report that targeting sphingosine kinase 2 (SphK2) by a selective inhibitor, ABC294640, significantly induces KSHV+ TIVE-LTC autophagic death. By using microarray analysis, we have identified the global gene profile affected by ABC294640 within KSHV+ TIVE-LTC and several novel “druggable” candidates closely related to cancer cell survival/growth. Finally, we found that targeting TIVE-LTC by ABC294640 effectively suppressed KSHV tumorigenicity by using a KS-like nude mice model. Overall design: TIVE-LTC were treated with vehicle control or SphK2 inhibitor ABC294640 (60 µM) for 24 h, and the gene expression signature was compared to respective vehicle controls
Project description:We compared the differentially expressed genes between the F9 Wt cells and F9 RAR gamma knock out cells before and after RA treatment. 3 replicates for each conditions. We also identified the RA responsive genes in the F9 Wt cells. Keywords: mutant type Overall design: F9 wt cells and F9 RAR gamma knock out cells are treated with RA (1 uM) or ethanol for 24 hours. 3 replicates for each condition.