Journal: The Journal of Neuroscience

Article Title: Neural Stem/Progenitor Cells Participate in the Regenerative Response to Perinatal Hypoxia/Ischemia

doi: 10.1523/JNEUROSCI.1898-05.2006

Figure Lengend Snippet: Primer sets used in RT-PCR

Article Snippet: Only those genes that were induced or repressed at least twofold in each replication are listed in supplemental (available at www.jneurosci.org as supplemental material ). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Transcript Primers EGFR (TaqMan) Applied Biosystems TaqMan Assay ID Rn00580398_m1 Notch1 (Lux) 5′-CAC GTA CTG CGA GCT GCC CTA CG[FAM]G-3′ 5′-GGC AGG TGC CTC CGT TCT-3′ 18S (TaqMan) Applied Biosystems TaqMan Assay ID Hs99999901_s1 18S (Lux) Invitrogen catalog #115HM-01 Open in a separate window Primer sets used in RT-PCR

Techniques: TaqMan Assay

Journal: The Journal of Neuroscience

Article Title: Neural Stem/Progenitor Cells Participate in the Regenerative Response to Perinatal Hypoxia/Ischemia

doi: 10.1523/JNEUROSCI.1898-05.2006

Figure Lengend Snippet: Signaling pathways involved in control of NSP fate are induced by perinatal H/I. Ipsilateral H/I and control hemispheres were dissected out after 48 h of recovery. A, Total RNA was isolated and amplified by qRT-PCR using primers specific for EGFR and Notch1 and normalized to expression of 18S. Values in parentheses indicate CV for the target gene in the sample groups; n = 10 for ipsilateral and contralateral conditions, and n = 4 for sham condition. *p < 0.05 versus sham; †p < 0.05 versus contralateral by pairwise fixed reallocation randomization. Immunostaining for Notch1 was performed on cryostat sections from H/I (B) and control (C) animals. In situ hybridization was performed on cryostat sections of contralateral (D, G), ipsilateral (E, H), and control (F, I) hemispheres using a digoxigenin-labeled RNA probe for Hes5 (D–F) and Hes1 (G–I). Arrows in E delineate the region of increased Hes5 expression. Inset in E shows high-magnification 100× Nomarski image of the Hes5+ region showing a subependymal Hes5+ cell body (arrowhead) with processes (arrow) projecting through the ependyma. There was no evident change in Hes1 expression. Scale bars: inset in E, 4 μm; F, 10 μm. cp, Choroid plexus; V, ventricle.

Article Snippet: Only those genes that were induced or repressed at least twofold in each replication are listed in supplemental (available at www.jneurosci.org as supplemental material ). table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Transcript Primers EGFR (TaqMan) Applied Biosystems TaqMan Assay ID Rn00580398_m1 Notch1 (Lux) 5′-CAC GTA CTG CGA GCT GCC CTA CG[FAM]G-3′ 5′-GGC AGG TGC CTC CGT TCT-3′ 18S (TaqMan) Applied Biosystems TaqMan Assay ID Hs99999901_s1 18S (Lux) Invitrogen catalog #115HM-01 Open in a separate window Primer sets used in RT-PCR

Techniques: Protein-Protein interactions, Control, Isolation, Amplification, Quantitative RT-PCR, Expressing, Immunostaining, In Situ Hybridization, Labeling

Journal: Nucleic Acids Research

Article Title: The Effects of Sequence Variation on Genome-wide NRF2 Binding—New Target Genes and Regulatory SNPs

doi: 10.1093/nar/gkw052

Figure Lengend Snippet: Regulatory loop of NRF2, miR-126 and KLF2 in HUVECs. ( A ) EGFL7 gene locus shown together with putative NRF2 binding sites (Class I: ‘Medium to Strong’ (relative binding 0.9–0.67); Class II: ‘Medium’ (relative binding 0.66–0.5); Class III: ‘Medium to Weak’ (relative binding 0.5–0.4); and Class IV: ‘Weak’ binding sites (relative binding 0.4–0.3)) and ENCODE HUVEC H3K27Ac data. miR-126 locus resides on intron 7. ( B ) HUVECs were treated with oxPAPC and NRF2 binding was measured with chromatin immunoprecipitation (ChIP) in 2, 4, 6 and 24 h time points (mean ± SD, n = 3). Results are shown as fold change against control samples. ( C ) Relative miRNA and host gene expression was measured with qPCR from oxPAPC-treated HUVECs in 4, 8, 12, 24 and 48 h time points (mean ± SD, n = 6). ( D and E ) Relative cellular expression and medium levels (exosomal and non-exosomal) for miR-126–3p and miR-126–5p were measured with qPCR from oxPAPC-treated HUVECs in 4, 8, 12, 16 and 24 h time points (mean ± SD, n = 3). ( F ) Regulatory loop for NRF2, miR-126 and KLF2. ( G ) The predicted binding of hsa-miR-126–3p to KLF2 mRNA (NM_016270.2 nt 466–487) ( H ) KLF2 expression was measured with qPCR from oxPAPC and control treated HUVECs after 8 h treatment. ( I and J ) HUVECs were transfected with inhibitor control, miR-126–3p inhibitor, mimic control and miR-126–3p mimic. Forty-eight hours after transfection, mimic samples were treated with oxPAPC to induce KLF2 expression. miR-126–3p and KLF2 expression were measured with qPCR (mean±SD, n = 3). ( K and L ) KLF2 and β-actin were measured from transfection samples after 16 h oxPAPC treatment. OxPAPC concentrations for (B–H) were 50 μg/ml and for (I–L) 20 μg/ml. In statistical analysis, samples were compared to respective control samples using unpaired t -test. * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: The cDNA templates were assayed in 10 μl PCR reactions with a LightCycler 480 Real-Time PCR System (Roche) according to the protocol of miRCURY LNA Universal RT miRNA PCR for miRNA samples and the protocol of Fast Start Universal Probe Master (Rox) (Roche) for mRNA samples using hsa-miR-126–5p (206 010, Exiqon) and -3p (204227, Exiqon) LNATM PCR primer set, UniRT and Assays-on-Demand target mixtures for KEAP1 (Hs00202227_m1, Applied Biosystems), Krüppel-like factor 2 ( KLF2 ) (Hs00360439_g1, Applied Biosystems) and EGFL7 (Hs00211952_m1, Applied Biosystems), and control genes PPIA1 (Hs04194521_s1, Applied Biosystems) and GAPDH (Hs99999905_m1, Applied Biosystems).

Techniques: Binding Assay, Chromatin Immunoprecipitation, Control, Gene Expression, Expressing, Transfection

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: EGF is required for cardiac differentiation of P19CL6 cells through interaction with GATA-4 in a time- and dose-dependent manner

doi: 10.1007/s00018-014-1795-9

Figure Lengend Snippet: EGF induces the dose- and time-dependent differentiation of P19CL6 cells into cardiomyocytes. a P19CL6 cells, with the specific induction of sarcomeric MHC proteins (detected by MF20 immunoblot) after exposure to 10 ng/ml EGF for 2 days (upper right) or 4 days (lower left), compared to the DMSO vehicle control (upper left) which did not result in detectable expression of sarcomeric MHC until day 16. b EGF-mediated induction of sarcomeric MHC proteins by MF20 immunoblot in P19CL6 cells upon the administration of 50 ng/ml EGF for 2 days (upper right) or 4 days (lower left). c Time-responsive induction of sarcomeric MHC proteins in P19CL6 cells by the administration of 100 ng/ml EGF for 2 days (upper right), 4 days (lower left), or 12 days (lower right). d Knocking down EGF (RNAi EGF) suppresses the induction of sarcomeric MHC proteins. The cell lysates were also subjected to immunoblot analysis on day 0 (lower). e Cardiac gene expression was assessed by real-time PCR in P19CL6 cells treated with 1 % DMSO. The results are the mean ± SEM. *p < 0.05 vs. control, and **p < 0.01 vs. control

Article Snippet: The GATA-4 RNAi and EGF RNAi constructs were designed according to the pSilencer neo™ Instruction Manual (Ambion, Foster, CA, USA) as described previously [ 46 ], and siRNA-negative control has no homology to any known mammalian gene.

Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: EGF is required for cardiac differentiation of P19CL6 cells through interaction with GATA-4 in a time- and dose-dependent manner

doi: 10.1007/s00018-014-1795-9

Figure Lengend Snippet: GATA-4 induces the differentiation of P19CL6 cells into cardiomyocytes. a The effect of GATA-4 on cardiomyocyte differentiation was assessed using MF20 immunoblotting in P19CL6 cell lysates from cells stably transfected with pCDNA3.1 (+) empty vector (upper), the GATA-4 expression vector, the GATA-4 knockdown vector, and Si RNA negative control vector (lower). The cells were cultured for 18 days in 1 % DMSO. At the indicated time points, the cell lysates were subjected to immunoblot analysis with the antibodies indicated in the figure. The cell lysates were also subjected to immunoblot analysis on day 0 (lowest, ctr control, G4 GATA-4, RiG4 RNAi-GATA-4, Ri ctr Si RNA control). b Cardiac gene expression was assessed by real-time PCR in P19CL6 cells treated with 1 % DMSO. On the indicated days, P19CL6 cells transfected with pcDNA3.1(+) empty vector were evaluated for α-MHC, GATA-4, and EGF mRNA levels. The results are the mean ± SEM. *p < 0.05 vs. control, and **p < 0.01 vs. control. c The mRNA levels of α-MHC, GATA-4, and EGF were analyzed on day 0 and days 4 to 18 in P19CL6 cells stably expressing GATA-4 during differentiation. The results are the mean ± SEM. *p < 0.05 vs. control, and **p < 0.01 vs. control. d The mRNA levels of α-MHC, GATA-4, and EGF were analyzed in P19CL6 cells with GATA-4 knock down at the indicated time points. The numbers in y axis stand for the activated number of target gene’s mRNA level corrected by S16. The results are the mean ± SEM. *p < 0.05 vs. control, and **p < 0.01 vs. control

Article Snippet: The GATA-4 RNAi and EGF RNAi constructs were designed according to the pSilencer neo™ Instruction Manual (Ambion, Foster, CA, USA) as described previously [ 46 ], and siRNA-negative control has no homology to any known mammalian gene.

Techniques: Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Expressing, Negative Control, Cell Culture, Real-time Polymerase Chain Reaction

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: EGF is required for cardiac differentiation of P19CL6 cells through interaction with GATA-4 in a time- and dose-dependent manner

doi: 10.1007/s00018-014-1795-9

Figure Lengend Snippet: The inhibition of cardiac differentiation by EGF RNAi or GATA-4 RNAi cannot be rescued by the overexpression of GATA-4 or by EGF treatment. a The expression of sarcomeric MHC was inhibited in P19CL6 cells over-expressing GATA-4 and the EGF RNAi cells (1), and in the GATA-4 RNAi cells exposed to 10 ng/ml EGF for 2 days (3) or 50 ng/ml EGF for 4 days (5). The cell lysates from the cells described above on day 0 were also subjected to immunoblot analysis (1 RNAi EGF + GATA-4, 2 Si RNA control + GATA-4, 3 RNAi GATA-4 + EGF 10 ng-2d, 4 Si RNA control + EGF 10 ng-2d, 5 RNAi GATA-4 + EGF 50 ng-4d, 6 Si RNA control + EGF 50 ng-4d). b–d α-MHC, GATA-4 and EGF mRNA levels were determined by real-time PCR analysis in P19CL6 cells treated as described in a. The results are the mean ± SEM of three independent experiments. *p < 0.05 vs. control, and **p < 0.01 vs. control

Article Snippet: The GATA-4 RNAi and EGF RNAi constructs were designed according to the pSilencer neo™ Instruction Manual (Ambion, Foster, CA, USA) as described previously [ 46 ], and siRNA-negative control has no homology to any known mammalian gene.

Techniques: Inhibition, Over Expression, Expressing, Western Blot, Real-time Polymerase Chain Reaction