Checkpoint kinase 1 is essential for fetal and adult hematopoiesis

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Checkpoint kinase 1 is essential for fetal and adult hematopoiesis

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Checkpoint kinase 1 is essential for fetal and adult hematopoiesis AThree independent Chk1fl/fl mT/mG (colored) and mT/mG (gray) Hoxb8‐FL progenitor cell lines were transduced with a retroviral construct carrying Cre (pMIP‐CRE‐IRES‐PURO) and monitored over time for GFP expression. Shown here are three technical replicates per genotype. Additionally, Chk1fl/fl mT/mG Hoxb8‐FL progenitor cells were FACS‐sorted for GFP+ and dsRED+ cells 48 h after transduction with the Cre‐construct. Sorted cells were processed for Western blotting using the indicated antibodies. BChk1fl/fl Hoxb8‐FL progenitor cells were transduced with a retroviral construct carrying Cre and GFP (pMIG‐CRE‐IRES‐GFP). Cells were FACS‐sorted 72 h after transduction with the retroviral construct and processed for Western blotting using the indicated antibodies. CPercentage of GFP+ cells indicative for CRE expression and recombination in the peripheral blood after tamoxifen administration by gavage (5 × 2 mg, 200 μl/mouse; daily). Bars represent means ± s.e.m. (Day 0 N = 9/10, WT/Chk1fl/fl, Day 7 N = 8/7, Day 14 N = 3/3). DQuantification of Chk1‐del PCR‐product enrichment in DNA from GFP+ vs. Tomato+ bone marrow cells sorted on day 8 after first TAM administration. Transgenic Cre levels were quantified in parallel for reference. Bars represent means ± s.e.m. from n = 5 mT/mG Vav‐CreERT2 Chk1fl/fl mice. E, F(E) Percentage of living LK or LSK cells and (F) percentage of GFP+ LK or LSK cells found in the bone marrow of mT/mG Vav‐CreERT2 (WT) and mT/mG Vav‐CreERT2 Chk1fl/fl (Chk1fl/fl) mice on day 8 or 15 after first TAM administration. Bars represent means ± s.e.m. (Day 8 N = 8/8, WT/Chk1fl/fl, Day 15 N = 7/8). GGFP+ (CRE) and Tomato+ (CRE−) total bone marrow cells were sorted on day 8 after first TAM administration and processed for Western blot using the indicated antibodies. Sorted cells from three animals—mT/mG Vav‐CreERT2 (WT) and mT/mG Vav‐CreERT2 Chk1fl/fl (Chk1fl/fl)—were pooled per lane. HThymocytes of the indicated genotypes were sorted based on GFP+ and Tomato+ on day 15 after the first TAM administration for Western analysis using the indicated antibodies. Data information: Asterisks indicate significant differences: *P < 0.05, **P < 0.01, ***P < 0.001 using unpaired Student's t‐test.Source data are available online for this figure. IF THIS IMAGE HAS BEEN PROVIDED BY OR IS OWNED BY A THIRD PARTY, AS INDICATED IN THE CAPTION LINE, THEN FURTHER PERMISSION MAY BE NEEDED BEFORE ANY FURTHER USE. PLEASE CONTACT WILEY'S PERMISSIONS DEPARTMENT ON PERMISSIONS@WILEY.COM OR USE THE RIGHTSLINK SERVICE BY CLICKING ON THE 'REQUEST PERMISSIONS' LINK ACCOMPANYING THIS ARTICLE. WILEY OR AUTHOR OWNED IMAGES MAY BE USED FOR NON-COMMERCIAL PURPOSES, SUBJECT TO PROPER CITATION OF THE ARTICLE, AUTHOR, AND PUBLISHER. EMBO Rep, Volume: 20, Issue: 8, First published: 17 June 2019, DOI: (10.15252/embr.201847026)