Journal: Poultry Science

Article Title: Multi-omics analysis reveals RBPJ-mediated regulation of EGF/ACTN2 / MYPN / COL21A1 in fibroblast during oviduct functional remodeling of duck

doi: 10.1016/j.psj.2026.106688

Figure Lengend Snippet: Identification of duck embryo fibroblasts and the effects of RBPJ overexpression on proliferation. (A) Immunofluorescence identification of decorin and vimentin in duck embryo fibroblasts. Target proteins (red, Alexa Fluor 488), nuclei (blue, Hochest 33342). Scale bar =100 μm. (B) Relative expression level of RBPJ mRNA, plot with Control as 1. (C) GAPDH and RBPJ protein band diagrams. (D) Quantified relative protein bands, plot with Control as 1. (E) Cell viability curve after RBPJ overexpression. (F) Staining maps of Group C and OE. (G) Quantitative plot of the proportion of EdU positive cells in two groups. pCD3.1 is the plasmid of group C and pCD3.1-RBPJ is the plasmid of group OE of RBPJ ( ⁎⁎ P < 0.01; ⁎⁎⁎ P < 0.001; ⁎⁎⁎⁎ P < 0.0001).

Article Snippet: The membrane was then incubated overnight at 4°C with the following primary antibodies diluted in Primary Antibody Dilution Buffer (P0023A, Beyotime, China): RBPJ (mouse monoclonal antibody, 1:2000, 66132-1-Ig, Proteintech, China), glyceraldehyde-3-phosphate dehydrogenase ( GAPDH , rabbit polyclonal antibody, 1:1000, 10494-1-AP, Proteintech, China) and β-tubulin (mouse monoclonal antibody,1:10000, 66240-1-Ig, Proteintech, China) (GAPDH and β-tubulin was used as the internal reference gene for normalization).

Techniques: Over Expression, Immunofluorescence, Expressing, Control, Staining, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: Exosomal hsa_circ_0004658 derived from RBPJ overexpressed-macrophages inhibits hepatocellular carcinoma progression via miR-499b-5p/JAM3

doi: 10.1038/s41419-021-04345-9

Figure Lengend Snippet: A Exosomes isolated from WT THP-1 derived macrophages (WT Mφ-Exo) and RBPJ-overexpressed macrophages (RBPJ +/+ Mφ-Exo) imaged by transmission electron microscopy (TEM) are approximately 100 nm in size. Scale bar = 100 nm. B Levels of exosome markers CD63, Alix, TSG101 and HSP70 in WT or RBPJ +/+ Mφ-Exo and WT or RBPJ +/+ Mφ (Cell) were determined by Western blotting. C , D Cell proliferation in HCC cell lines SMMC-7721 and HepG2 treated with exosomes derived from RBPJ-overexpressed macrophages was assessed by a CCK-8 assay. E Flow cytometry is performed to indicate cell apoptosis. The percentage of apoptosis was then measured. All experiments were performed three times. ** P < 0.01, as indicated.

Article Snippet: To obtain WT Mφ and RBPJ +/+ Mφ, THP-1 cells were transfected with the pCMV6 empty vector or pCMV6 overexpressing RBPJ (OriGene, Rockville, MD, USA) and seeded at a density of 0.5 × 10 6 cells per well in a six-well culture plate.

Techniques: Isolation, Derivative Assay, Transmission Assay, Electron Microscopy, Western Blot, CCK-8 Assay, Flow Cytometry

Journal: Cell Death & Disease

Article Title: Exosomal hsa_circ_0004658 derived from RBPJ overexpressed-macrophages inhibits hepatocellular carcinoma progression via miR-499b-5p/JAM3

doi: 10.1038/s41419-021-04345-9

Figure Lengend Snippet: To remove hsa_circ_0004658 from exosomes, siRNA of hsa_circ_0004658 was transfected into THP-1 cells and Mφ-Exo were collected at 48 h post-transfection (RBPJ +/+ Mφ-Exo-si-circRNA). HCC cell lines SMMC-7721 and HepG2 were cocultured with WT Mφ-Exo, RBPJ +/+ Mφ-Exo or RBPJ +/+ Mφ-Exo-si-circRNA (5 μg/ml). A , B Cell proliferation in HCC cell lines SMMC-7721 and HepG2 was assessed by a CCK-8 assay. C , D Cell migration in HCC cell lines SMMC-7721 and HepG2 was assessed by Transwell assay. E , F Cell apoptosis in HCC cell lines SMMC-7721 and HepG2 was assessed by flow cytometry. All experiments were performed three times. **, ## P < 0.01, as indicated. * vs. WT Mφ-Exo, # vs. RBPJ +/+ Mφ-Exo.

Article Snippet: To obtain WT Mφ and RBPJ +/+ Mφ, THP-1 cells were transfected with the pCMV6 empty vector or pCMV6 overexpressing RBPJ (OriGene, Rockville, MD, USA) and seeded at a density of 0.5 × 10 6 cells per well in a six-well culture plate.

Techniques: Transfection, CCK-8 Assay, Migration, Transwell Assay, Flow Cytometry

Journal: Nature Communications

Article Title: Direct reprogramming of fibroblasts into neural stem cells by single non-neural progenitor transcription factor Ptf1a

doi: 10.1038/s41467-018-05209-1

Figure Lengend Snippet: Ptf1a reprogramming activity depends on Notch-independent interaction with Rbpj. a Schematic depicting the trimeric DNA-binding complex formed among Ptf1a, Rbpj, and an E-protein, as well as the Ptf1a W298A C-terminal mutant that lacks the ability to interact with Rbpj. b Unlike Ptf1a lentivirus-infected MEFs, MEFs transduced with Ptf1a W298A lentiviruses failed to undergo morphological changes by day 6 or form neurospheres by day 9. c Quantification of neurospheres induced by Ptf1a and Ptf1a W298A . MEFs (4 × 10 4 ) were seeded into each well of 12-well plates, infected with Ptf1a or Ptf1a W298A viruses, and neurospheres in each well were then counted at day 10 and 14 following virus infection. There was a dramatic decrease of neurospheres in Ptf1a W298A -induced samples at day 10 and 14. Data are presented as mean ± SD ( n = 3). Asterisks indicate significance in unpaired two-tailed Student’s t -test: * P < 0.005, ** P < 0.0001. d MEFs infected with Ptf1a lentiviruses generated neurospheres at day 8 that were immunoreactive for Sox2, Pax6, and Nestin, whereas MEFs infected with Ptf1a W298A viruses were negative for these NSC markers. Cells were counterstained with nuclear DAPI. e Neurosphere formation by Ptf1a in MEFs infected with lentiviruses expressing Rbpj shRNA or scrambled Rbpj shRNA. f Relative expression levels of Rbpj in MEFs infected with lentiviruses expressing Rbpj shRNA or scrambled Rbpj shRNA as determined by qRT-PCR assay. Data are presented as mean ± SD ( n = 6). Asterisk indicates significance in unpaired two-tailed Student’s t -test: * P < 0.0001. g Quantification of neurospheres induced by Ptf1a in the presence of Rbpj shRNA or scrambled Rbpj shRNA. MEFs (4 × 10 4 ) were seeded into each well of 12-well plates, infected with Ptf1a lentiviruses and viruses expressing Rbpj shRNA or scrambled Rbpj shRNA. Neurospheres in each well were counted at day 10 following virus infection. Data are presented as mean ± SD ( n = 6). Asterisk indicates significance in unpaired two-tailed Student’s t -test: * P < 0.0005. Scale bars, 160 μm ( e ), 80 μm ( b ), and 40 μm ( d )

Article Snippet: For Rbpj knockdown assay, we purchased lentiviral vectors encoding mouse Rbpj short hairpin RNA (shRNA) (OriGene, TL512813) or scrambled Rbpj shRNA (OriGene, TL512813).

Techniques: Activity Assay, Binding Assay, Mutagenesis, Infection, Transduction, Two Tailed Test, Generated, Expressing, shRNA, Quantitative RT-PCR

Journal: Nature Communications

Article Title: iSuRe-Cre is a genetic tool to reliably induce and report Cre-dependent genetic modifications

doi: 10.1038/s41467-019-10239-4

Figure Lengend Snippet: iSuRe-Cre DNA construct, ES cells, and mice. a iSuRe-Cre DNA construct used to produce gene-targeted or transgenic ES cells and mice. ROSA26 hom., ROSA26 locus homology arms; INS, chicken β-globin HS4 insulator sequence; FRT, short DNA sequences recognized by the recombinase Flp allowing deletion of the PGK-Neo selection cassette; P1-P4, primers used to genotype ES cells or mice. For further abbreviations and their definitions see also Fig. legend. b PCR result with primers detecting the integration of the vector in the ROSA26 locus of ES cell clones 1–18. c PCR result with primers detecting the presence of the iSure-Cre allele in the genome of ES cells 1–18. d Representative images of different ROSA26-targeted (Gt(ROSA)26Sor) ES cell clones at baseline. N-PhiM is expressed if the allele is non-recombined, and MbTomato marks cells that had recombination of the construct without induction. Clone #6 has a higher proportion of non-recombined N-PhiM+ cells. e Representative images of different Tg(iSuRe-Cre ) ES cell clones at baseline. Clone #18 has a higher proportion of non-recombined N-PhiM+ cells. f Mouse chimeras generated with Gt(ROSA)26Sor-iSuRe-Cre ES cell clone #6 were interbred with Rbpj flox/flox animals, and the progeny were genotyped for the Rbpj and iSuRe-Cre alleles. All animals containing the iSuRe-Cre allele in the ROSA26 locus had deletion of the Rbpj -floxed allele and expressed the MbTomato reporter in all cells. g Mouse chimeras generated with Tg(iSuRe-Cre) ES cell clone #18 were interbred with Rbpj flox/flox animals, and the progeny were genotyped for the Rbpj and iSuRe-Cre alleles. Animals containing the Tg(iSuRe-Cre) allele did not have deletion of the Rbpj-floxed allele and did not express the MbTomato reporter. Instead they expressed the N-PhiM reporter due to the absence of Cre activity. Scale Bars 100 μm

Article Snippet: RNA was extracted according to the Qiagen protocol. cDNA was synthetized with the High Capacity cDNA kit from Applied Biosystems (AB). cDNA was pre-amplified with the Taqman PreAmp Master Mix (AB) and after qRT-PCR with gene-specific Taqman assays (Cdh5: Mm00486938_m1; Notch1: Mm00435245_m1; KDR: Mm00440085_m1; Rbpj:Mm01217627_g1; GAPDH: Mm99999915_g1) and universal master mix was performed on a AB 7900 qRT-PCR machine.

Techniques: Construct, Transgenic Assay, Sequencing, Selection, Plasmid Preparation, Clone Assay, Generated, Activity Assay

Journal: Nature Communications

Article Title: iSuRe-Cre is a genetic tool to reliably induce and report Cre-dependent genetic modifications

doi: 10.1038/s41467-019-10239-4

Figure Lengend Snippet: The Tg(iSuRe-Cre) allele increases the efficiency of inducible genetic modifications. a – c Representative confocal micrographs of P6 retina vessels labelled with IsolectinB4 (endothelial surface) and anti-ERG antibody (endothelial nuclei), obtained from animals with the genotype indicated to the left and induced with high-dose tamoxifen from P1 to P3. Images represent the observed phenotypic variability; EC number for each image is depicted in chart c (yellow dots). 20 vs 24 retina microscopic fields were analysed ( n = 5 vs n = 6 animals per group). d Linear regression showing the high correlation (r 2 ) between total Erg + EC number (phenotype) and the number of Erg + /MbTomato- cells. e , f Representative confocal micrographs of P6 retina vessels from animals with the genotype indicated to the left and induced with high-dose tamoxifen from P1 to P3. Images represent the phenotypic variability of animals with the same genotype. EC number and reporter expression frequency is indicated below the figures. A comparison of EC number and reporter expression frequency is depicted in chart for 24 vs 34 retina microscopic fields ( n = 6 animals per group). Yellow dots in chart f represent the values for images in b and e. g qRT-PCR analysis of Kdr mRNA levels from FACS-sorted liver ECs ( n = 5 animals), or immunostaining analysis of liver sections of animals ( n = 3) with the indicated genotype (see also Supplementary Fig. ). h Semi-quantitative competitive PCR showing the efficiency of Kdr, Rbpj and Nmyc deletion in FACS-sorted cells of adult mice with the indicated genotypes and induced with tamoxifen. Note that some cross-contamination of samples and DNA may occur during tissue dissociation and FACS of mutant and wild-type cells. i Semi-quantitative competitive PCR for the Notch1 floxed allele and a control genomic sequence showing the efficiency of the Ubc-CreERT2 induced Notch1 deletion in Tomato+ and Tomato- cells of the liver. j Semi-quantitative competitive PCR showing the efficiency of Rbpj gene inducible deletion in the Tomato− and Tomato+ cells of several distinct organs from Rosa26-CreERT2 mice induced with tamoxifen. k Epas1 mRNA relative levels (qRT-PCR) in LysM- Cre-reporter-expressing bone marrow-derived macrophages ( n = 4). Scale Bars 200 μm. Error bars indicate StDev; ** p < 0.001; *** p < 0.0001. Two-tailed unpaired t -test (4c) or ANOVA (4 g and 4k). Source data are provided as a Source Data file

Article Snippet: RNA was extracted according to the Qiagen protocol. cDNA was synthetized with the High Capacity cDNA kit from Applied Biosystems (AB). cDNA was pre-amplified with the Taqman PreAmp Master Mix (AB) and after qRT-PCR with gene-specific Taqman assays (Cdh5: Mm00486938_m1; Notch1: Mm00435245_m1; KDR: Mm00440085_m1; Rbpj:Mm01217627_g1; GAPDH: Mm99999915_g1) and universal master mix was performed on a AB 7900 qRT-PCR machine.

Techniques: Expressing, Comparison, Quantitative RT-PCR, Immunostaining, Mutagenesis, Control, Sequencing, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: iSuRe-Cre is a genetic tool to reliably induce and report Cre-dependent genetic modifications

doi: 10.1038/s41467-019-10239-4

Figure Lengend Snippet: The Tg(iSuRe-Cre) allele enables multiple gene deletions in single cells or tissues. a The schemes illustrate the Dll4 and Kdr floxed alleles, showing inter-LoxP-site genetic distance, which is significantly larger in the Kdr allele. All four alleles must be deleted to achieve full dual gene loss-of-function. Kdr and Dll4 proteins are expressed in most liver ECs (ERG+, nuclei) of Dll4 flox/flox /Kdr flox/flox animals injected with tamoxifen on 3 consecutive days. b Adult mice carrying in addition the Tg ( Cdh5-CreERT2) and Tg(iSuRe-Cre) alleles and treated with the same high-dose tamoxifen for 3 consecutive days show very pronounced deletion of Dll4 , but not Kdr , in liver MbTomato - /ERG + ECs (yellow arrowheads). However, MbTomato + cells (white arrowheads) have complete deletion of both genes. c Quantification of the immunostaining signals for ERG, Dll4, Kdr, and MbTomato in large liver sections of the indicated animals. d Illustration of the Myc , Mycn , and Rbpj-floxed alleles showing the genetic distances between the LoxP sites. e Genotypes of control and mutant adult mice injected once with 1 mg of tamoxifen and used for gene-deletion quantification by PCR. The control PCR band provides a DNA input quantitative control for the PCR, since it corresponds to a wild-type genomic sequence, present in all DNA samples (see supplementary table for primer sequences). Animals containing the Tg ( Cdh5-CreERT2) and Tg(iSuRe-Cre) alleles have deletion of the six floxed alleles only in FACS-sorted MbTomato + cells, as detected by semi-quantitative competitive PCR. Weak Mycn and Rbpj -floxed bands in the MbTomato + sample PCR may result from incomplete gene-deletion or contamination of this sample with MbTomato-negative cells, or their DNA, during the FACS protocol. f Image J quantification of the relative intensity of the floxed and control PCR gel bands shown in e , providing an estimate of the degree of the indicated floxed gene deletion. Scale Bars 65 μm. Error bars indicate StDev. ** p < 0.001. NS nonsignificant. One-way ANOVA with Tukey’s post hoc test. Source data are provided as a Source Data file

Article Snippet: RNA was extracted according to the Qiagen protocol. cDNA was synthetized with the High Capacity cDNA kit from Applied Biosystems (AB). cDNA was pre-amplified with the Taqman PreAmp Master Mix (AB) and after qRT-PCR with gene-specific Taqman assays (Cdh5: Mm00486938_m1; Notch1: Mm00435245_m1; KDR: Mm00440085_m1; Rbpj:Mm01217627_g1; GAPDH: Mm99999915_g1) and universal master mix was performed on a AB 7900 qRT-PCR machine.

Techniques: Injection, Immunostaining, Control, Mutagenesis, Sequencing