Single cell RNA sequencing of intestinal fibroblast-crypt co-cultures in organoid growth media with/without Ptger4/EP4 inhibitor (Drop-seq)
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ABSTRACT:
PROVIDER: PRJNA596992 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10745740_1.fastq.gz | Fastqsanger.gz | |||
| SRR10745740_2.fastq.gz | Fastqsanger.gz | |||
| SRR10745741_1.fastq.gz | Fastqsanger.gz | |||
| SRR10745741_2.fastq.gz | Fastqsanger.gz |
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Project description:Mouse small intestinal crypts were co-cultured with primary intestinal fibroblasts in Organoid Growth Media (OGM) with/without inhibitor for the prostaglandin E2 receptor Ptger4 (EP4), labeled respectively as “Ptger4-OFF” and “Ptger4-ON” conditions. Crypts co-cultured with fibroblasts in Ptger4-ON conditions (OGM containing DMSO vehicle control) acquired a spheroid morphology. Crypts co-cultured with fibroblasts in Ptger4-OFF conditions (OGM supplemented with Ptger4 inhibitor) developed into budding organoids. Fibroblasts were isolated from the small intestine of wild type mice. For the co-cultures 2 x 10e4 fibroblasts were seeded in 48-well plates overnight. Freshly isolated crypts (n = 500) were suspended in 1:1 Matrigel (Corning, 356231) and IntestiCult Organoid Growth Medium (Stem Cell Technologies, 06005) and added as an overlay on the fibroblasts. Crypts/fibroblasts were co-cultured with IntestiCult Organoid Growth Medium for 4 days. The ONO-AE3-208 Ptger4 (EP4) inhibitor (Cayman, 14522) dissolved in DMSO was added to the co-cultureson days 0 and 2 at a final concentration of 10 uM. DMSO was used as a vehicle control for the untreated co-cultures. On day 4, one pool of six Ptger4-ON co-cultures and one pool of six Ptger4-OFF co-cultures were subjected to the Drop-seq protocol.
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Project description:Recognition of foreign antigens by B cell receptor (BCR) on mature B cells leads to their clonal expansion, which is critically important for the effective host defence of the organism. However, excessive antigenic responses or reaction of B cells against bodyâs own components frequently lead to diverse immune diseases, such as B-cell lymphoma or autoimmunity, that often affect humans. Identification of genes that restrain uncontrolled proliferation of B cells is therefore an important goal towards understanding the origin of such diseases. Here we identify Ptger4 as a negative feedback regulator of B-cell proliferation in response to BCR triggering, and show that its encoded EP4 receptor is a principal molecule conveying the growth-suppressive effect of prostaglandin E2 (PGE2). In controlled in vitro assays, Ptger4-/- B cells showed augmented proliferative response and increased expression of activating genes upon BCR stimulation. Stable knock-down of Ptger4 in B-cell lymphoma markedly accelerated tumour spread in mice, while Ptger4 overexpression yielded significant protection. Lack of Ptger4 rendered mouse B cells completely resistant to proliferation arrest signalled by PGE2, and we find by transcriptional profiling that intrinsic activity of Ptger4 and PGE2-EP4 signalling target a similar set of activating genes. We further show that Ptger4 inhibits mouse B-cell activation in vivo and find it significantly downregulated in human B-cell lymphoma. Our results demonstrate that Ptger4 functions in B cells as a candidate tumour suppressor whose activity is regulated by the presence of PGE2 in the microenvironment. These findings suggest that targeting EP4 receptor for prostaglandin may present a novel strategy for treatment of B-cell diseases. Keywords: time course, cell type comparison, compound treatment design, microarray experiment record B cells were extracted from spleen of Ptger4+/+ and Ptger4-/- mice, and incubated in vitro for the indicated period with or without anti-IgM (Fab')2 antibody fragments and co-treated with or without PGE2. Total RNA was extracted, amplified, labelled with Cy3 or Cy5, and hybridized to mouse 24k oligo-arrays using a dye-swap strategy.
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