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Collection : Elife (E-Journal - Via Crossref)

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Figure 3—figure supplement 1. Knockdown of CCN1 decreased tip cell markers. ; (A) After starved for 16 hr, HUVECs were treated with serum for 10, 30 and 60 min and lysed for Western blot analysis against CCN1. (B) After transfection with siVEGF, starved HUVECs were treated with CCN1 (10 ng/ml) for 1 hr and lysed for Western blot analysis. β–actin was used for loading control. (C) After transfection of two small interfering RNA of CCN1 in HUVECs, starved HUVECs were treated with CCN1 (10 ng/ml) for 1 hr and mRNA expression of CCN1, VEGFR2, DLL4 was measured by semiquantitative RT-PCR. β–actin was used for loading control. (D) Spheroid assays were performed with siCCN1 transfected HUVECs in the presence or absence of serum for 48 hr *p

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Figure 6—source data 1. TMEM63C expression in human podocytes using small interfering RNA (siRNA) methodology.

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Figure 4. SCP1 inhibited AKT-mediated angiogenesis ; (A) SCP1 deletion promoted VEGF-induced AKT activation in HUVECs. HUVECs were transfected with siNC (Small Interfering RNA for Normal Control) or siSCP1 (Small Interfering RNA for SCP1) for 72 h and stimulated with VEGF (100 ng/ml) as indicated after starvation for 8 h. (B) SCP1 impaired HUVEC tube formation through AKT. HUVECs were overexpressed with SCP1 with or without AKT-S473D. The cells were placed in plates coated with Matrigel and tubular structures were photographed after 6 h. The tube lengths were measured in each field. *p

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Figure 1. Molecular analysis of the POI family. ; (A) Pedigree of the Finnish family with two sisters affected with POI. (B) Exome data visualization in IGV (Integrative Genomics Viewer) shows a high coverage of the variant position and a large number of reads in the three individuals. (C) Sanger sequencing confirmed the presence of the variant at a homozygous state in both affected sisters and at a heterozygous state in both parents and their brother. (D) Structure of the FANCM gene and protein, and position of the causal variant. (E) Western blots of FANCM in the POI family. HEK293 cell transfected with a FANCM-specific siRNA were used to validate the specificity of the anti-FANCM antibody. To transiently deplete FANCM, HEK293 cells were transfected with 20 nmol/L of small interfering RNA (siRNA) targeting FANCM, 5'-GGC-UAC-GUC-CAG-GAG-CGC-3' with the CaCL2 method. Panel present the result of a representative experiment on at least three independent analysis.

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Table 2. Design of small interfering RNA output for the bab2 ORF.

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Table 1. Design of small interfering RNA output for the bab1 ORF.

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Figure 4—figure supplement 2. Knock-down efficiency and specificity of small interfering RNA in HD patient fibroblasts (Q45). ; Cells grown in 35 mm culture dishes were treated with 800 pmoles of On-target plus smartpool SiRNA for 72 hr. Cells were lysed and immunoblotted with antibodies for PIP4Kα, PIP4Kβ, PIP4Kγ and GADPH.

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Figure 5. GABPα co-localizes with P300 and modulates ZRS acetylation status. ; (A) Chromatin immunoprecipitation (ChIP) analysis from two biological replicates on the 14Fp cell line using GABPα and p300 antibodies using tiling microarrays. Summary is presented using two different genomic regions, the y axis is log2 for each ChIP/input DNA and the x axis represents a segment of DNA from the microarray. The DNA region containing the ZRS is highlighted by the grey shading. As controls, the whole of the Shh coding region plus promoter is shown. (B) 14Fp nuclear cell extracts from cells stably transfected with 3Xflag-Gabpα (Figure 5—figure supplement 1E) treated with or without doxycycline were analysed by immunoprecipitation with anti-p300 antibody followed by Western blot analysis with anti-flag-tag antibody. As control the empty vector plus doxycycline was used. (C) Western blot analysis with anti-GABPα of 14Fp nuclear cell extracts transiently transfected with Gabpα small interfering RNA (siRNA) (siGabp-α) or nonspecific siRNA (siCTR) and trichostatin A (TSA) treated. (D) Quantitative reverse transcriptase (qRT)-PCR was used to detect the messenger RNA (mRNA) levels of Shh (grey box) and Gabpα (black box) in 14Fp cells transfected with Gabpα siRNA or nonspecific siRNA. Eighteen hours after transfection, the cells were treated with 1 μM TSA for 24 hr. Shh and Gabpα levels were evaluated relative to control and normalized to glyceraldehyde 3-phosphate dehydrogenase levels from two biological replicates. (E) qRT-PCR to detect the mRNA levels of Shh (black box) and Gabpα (grey box) in 14Fp cells stably transfected with 3Xflag-Gabpα vector and an empty vector as control. Data points represent the mean ± SEM of three biological replicate. (F) Chromatin from 14Fp cells stably transfected with 3Xflag-Gabpα vector and an empty vector as control was analysed by ChIP for H3K27ac histone modification and ETV4 enrichment. DNA was quantified by q-PCR using the ZRS 5’ spatiotemporal (5’ST) activity oligo set. Data are represented as mean ± SEM of the fold enrichment over nonspecific IgG recoveries from two independent experiments. (G–H) Chromatin from 14Fp cells stably transfected with 3Xflag-Gabpα vector and an empty vector as control was analysed by ChIP for GABPα and P300. DNA was quantified by q-PCR using the ZRS 3’ long-range (3’LR) and 5’ST activity oligos sets. Average of percentage of input ±SEM from two independent experiments are plotted.

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Figure 6. ETV4 acts as a repressor via interactions with HDAC2. ; (A) Quantitative reverse transcriptase (q RT)-PCR to detect the messenger RNA (mRNA) levels of Shh (black box) in 14Fp cells transfected with HDAC1 and HDAC2 small interfering RNA (siRNA) either alone or combined and with nonspecific siRNA as control. Data were collected after 18 hr of transfection. Shh levels were evaluated relative to control and normalized to glyceraldehyde 3-phosphate dehydrogenase levels. Data points represent the average of triplicate determinations ± SEM. (B) ChIP from two biological replicates using the 14Fp cell line and anti-ETV4 and HDAC2 antibodies analysed by hybridizing to tiling microarrays (Figure 5—figure supplement 1B). Summary is presented using two different genomic regions, the y axis is log2 for each ChIP/input DNA and the x axis represents a segment of DNA from the microarray. The DNA region containing the ZRS is highlighted by the grey shading. As controls, the whole of the Shh coding region plus promoter is shown. (C) 14Fp nuclear cell extracts were analysed by immunoprecipitation with anti-ETV4 and IgG antibodies followed by Western blot analysis with anti-HDAC2. (D) qRT-PCR to detect the mRNA levels of Shh (black box) and Etv4 (grey box) in 14Fp cells transiently transfected with ETV4 siRNA (siRNA Etv4) or nonspecific siRNA (siRNA Ctr). Data points represent the average of triplicate determinations ± SEM. (E) Shown are results from chromatin immunoprecipitation (ChIP) analysis using anti-HDAC2 and ETV4 antibody after 24 hr of trichostatin A (TSA) treatment. Recovered DNA sequences were quantified by quantitative PCR using 5’ spatiotemporal (5’ST) oligo set. Average percentage of input and ±SEM from two independent experiments are plotted. The IgG did not give detectable signal.

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Figure 9. 3’ mismatches detected in 24 nt siRNAs and Pol IV/RDR2-dependent RNAs (P4R2 RNAs) may reflect RDR2 terminal transferase activity. ; (A) Genome browser view of P4R2 RNAs (shades of blue) and 24 nt small interfering RNA (siRNAs) (shades of gray) at a representative locus, an AtSN1 retrotransposon on chromosome 3. Each horizontal bar represents a specific RNA sequence (RNA-seq), with arrows depicting their direction relative to the Arabidopsis reference genome sequence (TAIR10). The intensity of shading reflects the abundance of each RNA species in the RNA-seq dataset. Brightly colored nucleotides, color coded for A, G, C, or U (see inset), represent nucleotides that do not match the corresponding DNA sequence of the locus. The dotted line highlights the coincident 5’ ends of the most abundant P4R2 RNAs at the locus (colored deep purple) and the most abundant siRNAs (colored black). (B) Heat map depicting the frequency of mismatched nucleotides at each position of RNAs ranging in size from 15 to 76 nt in dcl2 dcl3 dcl4 triple mutant plants. To correct for the frequency of errors inherent to sequencing, mismatch values for each position of 15–76 nt RNAs in wild-type plants were subtracted prior to plotting the data. Only read sequences with single mismatches or perfect matches to the reference genome were utilized for this analysis. (C) Over-expression and purification of recombinant RDR2. The image on the left shows a 7.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) gel, stained with Coomassie blue, showing molecular weight markers (M), proteins of un-infected High Five cells (lane 1), proteins of High Five cells 72 hr after infection with baculovirus expressing recombinant RNA-dependent RNA polymerase 2 (RDR2) (lane 2), and purified recombinant V5-tagged RDR2 after affinity purification and elution with V5 peptide. The image at right shows anti-RDR2 and anti-V5 immunoblots of the same three protein samples. For RDR2 detection, rabbit anti-RDR2 primary antibody was used in conjunction with donkey anti-rabbit HRP-conjugated secondary antibody. Detection of V5-tagged RDR2 involved anti-V5 HRP conjugate antibody. (D) RDR2 terminal transferase activity. Recombinant RDR2 or an active-site mutant form of RDR2 (RDR2-ASM) was incubated with alpha-labeled 32P-CTP and 51 nt RNA substrates bearing 3’ hydroxyl or 3’ dideoxy termini. Reaction products were subjected to denaturing polyacrylamide gel electrophoresis (PAGE) and autoradiography. For gel lane 4, reaction products were treated with RNase One, which degrades single-stranded RNAs, prior to PAGE. RNA size markers were run in lane M. The 51 nt RNA template, 5’ end-labeled using T4 polynucleotide kinase, was run as a size marker in the lane at far right.

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