Fig 1.
Expression of Ankle1 in murine tissues.
(A) Representative end point PCR from tissue cDNA, Actin and Gapdh serve as loading control; (B) Quantitative real time PCR, samples are normalized to Ankle1 expression in primary mouse embryonic fibroblasts (MEFs), 3 ≤ n ≤ 5, error bars: standard deviation.
Fig 2.
(A) Schematic drawing of the Ankle1 wild-type locus with exons 1 to 9, the knock-out allele carrying the complete deletion cassette (Ankle1-) consisting of FRT recombination sites (semi circle), splice acceptors site (En2 SA), Internal Ribosome Entry Site (IRES), LacZ gene (LacZ), polyadenylation site (pA), loxP recombination site (triangle), β-actin promoter (β -act.-p.) and aminoglycoside phosphotransferase gene (neo) and the knock-out allele after CRE-recombination (Ankle1Δneo) carrying the reporter gene cassette only and the knock-out allele after FLP-mediated recombination (Ankle1Δ); (B) Birth statistics of different Ankle1-deficient mouse strains (for organization of the genomic locus see Fig 2A) from heterozygous pairings, for determination of statistical significant differences between the observed and the expected birth ratios (25% wildtype, 50% heterozygous, 25% knock-out) a Chi square test was performed, p≤0.05 statistical significant, p≤0.01 very statistical significant; (C) Weight of Ankle1+/+ and Ankle1Δ/Δ animals 1–4 months and 4–8 months old, paired students t-test, statistical significance p≤0.05, 17 ≤ n ≤ 19; (D) Survival curve for Ankle1+/+ and Ankle1Δ/Δ mice, log rank (Mantel-Cox) test p = 0.11, n = 18.
Fig 3.
Flow cytometric analysis of early hematopoietic development in 9–10 months old Ankle1Δ/Δ mice.
(A) Representative dot plot of lineage negative bone marrow cells (BMCs) of an Ankle1+/+ and an Ankle1Δ/Δ mouse and relative quantification of the KLS cells (c-Kit+ and Sca-1+, boxed) depicted in a scatter plot; (B) Representative dot plot of c-Kit+ Lin- IL7Rα- Sca-1- BMCs of an Ankle1+/+ and an Ankle1Δ/Δ mouse and relative quantification of the boxed, CD16/32low CD34+ common myeloid progenitors (CMPs) in a scatter plot; (C) Representative dot plot of IL7Rα+ Lin- BMC of an Ankle1+/+ and an Ankle1Δ/Δ mouse and relative quantification of the c-Kit+ Sca-1+, common lymphoid progenitors (CLPs) depicted in a scatter plot; scatter plots indicate the mean (n = 5), error bars: standard deviation, paired students t test was used for determination of statistical significant differences (p≤0.05).
Fig 4.
Flow cytometric analysis of lymphoid differentiation in 8 weeks old Ankle1Δ/Δ mice.
(A) Representative dot plots of B cell progenitors (B220+ IgM-) and B cells (B220+ IgM+) in white blood cells (WBC) from bone marrow of an Ankle1+/+ and an Ankle1Δ/Δ mouse and relative quantification of the B220+ IgM- and B220+ IgM+ cells in a bar graph; (B) Representative dot plots of consecutive developmental stages of T cell progenitors in thymus of an Ankle1+/+ and an Ankle1Δ/Δ mouse and its relative quantification; Relative quantification of (C) B220+ and (D) CD4+ and CD8+ WBCs in spleen; error bars: standard deviation, paired students t test was used for determination of statistical significant differences (p≤0.05), n = 5.
Fig 5.
Flow cytometric analysis of erythroid differentiation in Ankle1Δ/Δ mice.
Cells were labeled with antibodies against Ter119 and CD71 (A) Representative dot plots of fetal liver cells from Ankle1+/+ and Ankle1Δ/Δ embryos (E 13.5), regions 1–5 (R1–R5) represent five erythroid maturation stages, bar graphs show the percentage of labeled cells in each differentiation stage, n litter = 3; Representative dot plots of (B) Bone marrow and (C) Splenic cells from an 9–10 months old Ankle1+/+ and an Ankle1Δ/Δ mouse, regions 1–4 (R1–R4) represent four erythroid maturation stages, bar graphs show the percentage of labeled cells in each differentiation stage, n = 5; error bars: standard deviation, students t test was used for determination of statistical significant differences (p≤0.05).
Fig 6.
Growth and DNA damage sensitivity of Ankle1-deficient mouse embryonic fibroblasts (MEFs).
(A) Growth curve of primary MEFs (pMEFs), n = 3, error bars: standard deviation; (B-D) Survival of pMEFs after chronic exposure to indicated DNA damaging agents for 72 h, n = 3, error bars: standard deviation; (E) Survival of pMEFs 72 h after single exposure to increasing doses of UV-light, n = 3, error bars: standard deviation; (F-I) Clonogenic survival assay of immortalized MEFs (iMEFs) after chronic exposure to indicated DNA damaging agents for 10–14 days, n = 2–3, error bars: standard error of the mean.
Fig 7.
Involvement of ANKLE1 in Holliday junction resolution.
(A) mRNA expression profile of Ankle1 and known proteins involved in Holliday junction resolution after double strand break repair in somatic cells, representative end point PCR, Actin and Gapdh serve as loading control; (B) Western blot and qPCR analysis of iMEFs 48 h after transfection with indicated siRNAs; Scatter plot of sister chromatid exchange (SCE) frequencies in (C) Wild-type iMEFs and (D) Wild-type vs. Ankle1Δ/Δ iMEFs following siRNA treatment, each dot represents one metaphase spread, horizontal bars represent the mean, error bars: standard error of the mean, p values were determined using the students t test, n.s. not significant, * significant (p≤0.05), ** very significant (p≤0.01), *** extremely significant (p≤0.001).