Fig 1: Mapping the genomic duplication in initial floxed Poldip2 mice.Two samples of genomic DNA were selected to map the duplication: one wild-type (WT) and one homozygous (Hom) floxed Poldip2, identified by copy number variation assays as in Fig 3A. Multiple primer pairs, annealing to chromosome 11 (Chr11) on either side of the Poldip2 gene, were designed. A: Validation of the qPCR method with SYBR green detection. The Hom sample amplified earlier than the WT with primer pairs annealing either 28 kb upstream (-28) or 52 kb downstream (52) of exon 4 in Poldip2, as expected for duplicated DNA. In contrast, both samples amplified at the same cycle number with a primer pair annealing within the leptin receptor (Lepr) on chromosome 4. Each amplification curve represents the average fluorescent intensity from 4–6 replicates. B: Mapping was carried out in five successive rounds of primer design and testing. In this schematic (not to scale), primer pairs are represented by facing arrowheads labeled according to the approximate distance in kb between their annealing site and floxed Poldip2 (in red at the center). Blue and green colors indicate primer pairs found to anneal inside and outside the duplication, respectively. In the first two rounds of screening, primer pairs were designed to anneal progressively further away from Poldip2 on Chr11. After finding primers annealing outside the duplication, the last three rounds progressively identified primers closer to the edges of the duplication. In the end, these results indicate that the length of the duplicated segment is between 304 kb (133+171) and 306 kb (134+172). C: Genomic map of duplicated DNA segment in floxed Poldip2 mice. The largest protein coding genes and their orientations are represented as thick arrows. Gene information and nucleotide numbering on Chr11 are from NCBI’s mouse genome (GRCm38.p4). Poldip2 is shown in red and truncated genes that overlap the edges of the duplication are shown in green.
Fig 2: TNFα partially reverses the effect of Poldip2 depletion. Poldip2+/+ and Poldip2+/− mice subjected to tMCAO received an intraperitoneal injection with or without 5 μg TNFα, immediately after tMCAO, followed by a 24 h reperfusion. Evans blue extravasation was measured as in Fig. 2. Bars represent means ± SEM from five to seven mice per group. Two-way ANOVA *p < 0.05 vs. Poldip2+/+ no TNFα; §p < 0.05 vs. Poldip2+/+ no TNFα; #p < 0.05 vs. Poldip2+/+ with TNFα and & p < 0.05 vs. Poldip2+/− no TNFα
Fig 3: Effect of Poldip2 siRNA on the expression level of downstream proteins and GFAP at 12 hours after BM induction. A, Representative Western blotting bands of Poldip2, MMPs, ß-DG, ZO-1, occludin, claudin-5, and GFAP. B-I, Quantitative analysis of Poldip2, MMPs, ß-DG, ZO-1, occludin, claudin-5, and GFAP expression levels. Data are presented as mean ± SEM, n = 6 per group. *P < .05 vs. sham group; & P < .05 vs. vehicle group; # P < .05 vs. scramble siRNA group
Fig 4: Initial floxed Poldip2 mice carry a genomic duplication.A: Copy number variation assays were performed by qPCR using two primer pairs and two TaqMan probes in each well to amplify both a reference gene and one short genomic segment at the indicated location (top). Bars are averages from quadruplicate representative samples ± confidence intervals calculated by CopyCaller software. Individual mouse ID numbers are indicated below each bar. WT is a wild type control and Em is an embryo. Apparent genotypes, obtained by conventional PCR as in Fig 2, are indicated at the bottom. The presence of 3 and 4 copies per genome is consistent with a duplication carried by heterozygotes (Het) and homozygotes, respectively. B: Increased expression of Poldip2 and neighboring genes in floxed mice. Poldip2 genotypes were determined by conventional PCR as in Fig 2, in addition to copy number variation assays, as in panel A, assuming that homozygotes (Hom) have 4 copies per genome. Total RNA was purified from duodenum. Expression of Poldip2 and adjacent genes Tmem199 and Tnfaip1, as well Nox4, a distant gene used as a control, was assessed by RT-qPCR and normalized to the wild type for each gene. Bars represent average ± SEM (n = 3–5 mice) **P< 0.01, ***P<0.001 vs. WT.
Fig 5: FAK activity mediates TNF-induced VCAM-1 upregulation. Seventy-80% confluent RBMECs were transfected with siControl or siPoldip2 and the following day transduced with AdFAK or AdLacZ (control for AdFAK). Two days after transduction, when cells had reached 100% confluence, RBMECs were stimulated with TNF (10 ng/ml). Six hours later, whole cell lysates were prepared and immunoblotted for VCAM-1, total FAK, Poldip2 and β-tubulin. Down-regulation of Poldip2 after siRNA knockdown as well as overexpression of FAK were verified by western blotting. (A) Representative VCAM-1, total FAK, Poldip2 and β-tubulin western blots. (B) Bars represent means ± SEM of 4–5 independent experiments normalized to β-tubulin. Two-way ANOVA *P < 0.05 vs. AdLacZ siPoldip2 TNF. ns not significant.
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