Whole transcriptome profiling of the steady state and Hh-treated (8 hrs) Alpk1+/- (heterozygous) and Alpk1-/- (knockout) BMDMs using RNA-Sequencing approach
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ABSTRACT:
PROVIDER: PRJEB27929 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR2713070_1.fastq.gz | Fastqsanger.gz | |||
| ERR2713070_2.fastq.gz | Fastqsanger.gz | |||
| ERR2713071_1.fastq.gz | Fastqsanger.gz | |||
| ERR2713071_2.fastq.gz | Fastqsanger.gz | |||
| ERR2713072_1.fastq.gz | Fastqsanger.gz |
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Project description:Alpk1-deficient mice demonstrate exacerbated colitis and increased IL-12/Th1 response upon challenge with an intestinal pathobiont, Helicobacter hepaticus (Hh). Hematopoietic compartment is driving the pathogenic phenotype in this animal model, and Alpk1 is highly expressed in myeloid cells (macrophages and dendritic cells). Alpk1 deficiency has a recessive phenotype, since Alpk1+/- (heterozygous) mice show the same phenotype as the wild type mice. Mouse bone-marrow derived macrophages (BMDMs) show elevated IL-12 production in Alpk1-/- mice in response to stimulation with Hh. Since the molecular mechanism of how Alpk1 deficiency affects macrophage response to Hh is unknown, we aimed to characterise global changes in gene expression in Alpk1+/- vs Alpk1-/- bone-marrow differentiated cells (BMDMs). Cells were isolated from bone marrow of Alpk1+/- and Alpk1-/- (mixture of bone marrows from three mice per genotype) and plated in BMDM differentiation medium (RPMI, 10% FCS, penicillin and streptomycin, 50 micro beta-mercapthoethanol, 20 ng/ml recombinant mouse GM-CSF(Peprotech)), 7 million cells in per 10 sm uncoated TC dish in 10 ml of medium for eight days, extra 10 ml of medium was added to plates at day 4, before collection and replating in 96-well plates, 150 thousand cells/200 microliters of differentiation medium per well in technical triplicates per genotype/stimulation condition (R1-R3 labels of the samples). The following day BMDMs were stimulated with MOI of 10 of Hh and 10ng/ml of mouse IFNg (Peprotech) (Alpk1+/- BMDMs – het _Hh_8h vs Alpk1-/- BMDMs – alpk1_Hh_8h) or IFNg only (het _nonstim_8h vs alpk1_nonstim_8h) before lysis for RNA extraction using Quick-RNA kit from Zymo Research. Purified RNA was submitted to the Welcome Trust Centre for Human Genetics (Oxford) for RNA-Sequencing
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Project description:Whole transcriptome profiling of the steady state and Hh-treated (8 hrs) Alpk1+/- (heterozygous) and Alpk1-/- (knockout) BMDMs using RNA-Sequencing approach
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Project description:Transcriptomic analysis of ALPK1-dependent responses in BMDMs from WT and ALPK1 knockout C57BL/6 mice upon ADP-Hep stimulation
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Project description:Bone marrow was harvested from Rosa26CreER; Stk40+/+ (WT; n = 3) and Rosa26CreER; Stk40loxp/loxp (Stk40 KO; n = 3) mice and differentiated for 6 days in the presence of 100 nM 4-OHT to generate WT and Stk40 KO bone-marrow derived macrophages (BMDMs). 2. On day 7 following differentiation BMDMs were treated with 100 ng x ml-1 LPS and harvested at 0 hrs, 6 hrs, 16 hrs, and 32 hrs following LPS exposure. 3. The cells were snap-frozen at the time of harvest. RNA was extracted using the Qiagen RNeasy mini kit as per manufacturer’s protocol including the on-column DNase digestion. Groups: There are cells from 3 mice x 2 genotypes x 4 time points G1: WT 0 hr LPS G2: WT 6 hr LPS G3: WT 16 hr LPS G4: WT 32 hr LPS G5: Stk40 KO 0 hr LPS G6: Stk40 KO 6 hr LPS G7: Stk40 KO 16 hr LPS G8: Stk40 KO 32 hr LPS
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Project description:Cancers are heterogeneous by nature. While traditional oncology screens commonly use a single endpoint of cell viability, altering the phenotype of tumor-initiating cells may reveal alternative targets that regulate cellular growth by processes other than apoptosis or cell division. We evaluated the impact of knocking down expression of 420 kinases in bi-lineage triple-negative breast cancer (TNBC) cells that express characteristics of both myoepithelial and luminal cells. Knockdown of ERN1 or ALPK1 induces bi-lineage MDA-MB-468 cells to lose the myoepithelial marker keratin 5 but not the luminal markers keratin 8 and GATA3. In addition, these cells exhibit increased β-casein production. These changes are associated with decreased proliferation and clonogenicity in spheroid cultures and anchorage-independent growth assays. Confirmation of these assays was completed in vivo, where ERN1- or ALPK1-deficient TNBC cells are less tumorigenic. Finally, treatment with K252a, a kinase inhibitor active on ERN1, similarly impairs anchorage-independent growth of multiple breast cancer cell lines. This study supports the strategy to identify new molecular targets for types of cancer driven by cells that retain some capacity for normal differentiation to a non-tumorigenic phenotype. ERN1 and ALPK1 are potential targets for therapeutic development.
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