Journal: Genomics

Article Title: Hypoxic environment promotes angiogenesis and bone bridge formation by activating Notch/RBPJ signaling pathway in HUVECs.

doi: 10.1016/j.ygeno.2024.110838

Figure Lengend Snippet: Fig. 5. Notch/RBPJ pathway in HUVECs induces osteogenic differentiation of BMSCs to form bone bridges under ischemia-hypoxia. (A)(B) BMSCs were induced by the supernatant of the HUVEC culture medium to differentiate into osteoblasts. Osteogenic markers were detected using RT-qPCR and Western blotting. Mean ± SD is indicated; * P ≤0.05. (C) ALP and ARS staining of BMSCs after subgroup culture. (D) Pathological sections and H&E staining of rat femoral growth plates after surgically adding different preparations of hemostatic sponges and blank controls (×100). The green dotted line shows new bone tissue. The black arrow shows the new blood vessels. (E) Micro-CT was taken from bilateral femurs of rats containing different hemostatic sponges (0.1 mL PBS or 0.1 mL of 10 mM/L Notch/RBPJ pathway activator or inhibitor) and blank control group to detect angulation deformity. Activator: 26◦, inhibitor: 5◦, PBS: 12◦, sham- operated: 0◦.

Article Snippet: 4) In the inhibitor group, rats were treated with left epiphyseal plate resection and implanted with a hemostatic sponge containing 0.1 mL of 10 mM/L Notch/RBPJ pathway inhibitor (RBPJ Inhibition-1, RIN1, MedChemExpress China).

Techniques: Quantitative RT-PCR, Western Blot, Staining, Micro-CT, Control

Journal: Cell Death & Disease

Article Title: The RBPJ/DAPK3/UBE3A signaling axis induces PBRM1 degradation to modulate the sensitivity of renal cell carcinoma to CDK4/6 inhibitors

doi: 10.1038/s41419-022-04760-6

Figure Lengend Snippet: a Bioinformatic analysis of the potential binding proteins of the promoter of DAPK3 via analysis the ChIP-seq datasets. b , c 786-O and ACHN cells were transfected with indicated plasmids for 24 h. Cells were harvested for western blotting analysis ( b ) and RT-qPCR analysis ( c ). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with three replicates ( n = 3). Ns not significant; ** P < 0.01; *** P < 0.001. d , e 786-O and ACHN cells were infected with indicated shRNAs for 72 h. Cells were harvested for western blotting analysis ( d ) and RT-qPCR analysis ( e ). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with three replicates ( n = 3). ** P < 0.01. f , g 786-O cells were treated with 0, 1 µM, 5 µM, and 10 µM the RBPJ inhibitors (the DAPK3 inhibitor) for 24 h. The WCL of cells were subjected to western blotting analysis ( f ) and RT-qPCR analysis ( g ). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with three replicates ( n = 3). Ns not significant; *** P < 0.001. h the ChIP-seq of RBPJ on the promoter region of DAPK3 . i 786-O cells were infected with indicated shRNAs for 72 h. Cells were harvested for ChIP-qPCR analysis by using the IgG or RBPJ antibodies. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with three replicates ( n = 3). ** P < 0.01; *** P < 0.001. j 786-O cells were transfected with indicated plasmids for 24 h. Cells were harvested for ChIP-qPCR analysis by using the IgG or RBPJ antibodies. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparisons test. Data presented as Mean ± SEM with three replicates ( n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001. k The correlation between RBPJ and DAPK3 were analyzed by the GEPIA web tool ( http://gepia.cancer-pku.cn/ ) in different types of cancer. l 786-O cells were infected with indicated shRNAs for 72 h. Cells were harvested for western blotting analysis. m 786-O cells were infected with indicated shRNAs for 48 h. Then, cells were treated with or without the 5 µM RBPJ inhibitor for another 24 h. Cells were harvested for western blotting analysis.

Article Snippet: The antibodies used as follows: UBE3A (10344-1-AP, Proteintech; 1:1000 dilution), PBRM1 (12563-1-AP, Proteintech; 1:500 dilution), DAPK3 (2928, Cell signaling technology, 1:1000 dilution), RBPJ (14613-1-AP, Proteintech; 1:1000 dilution); P21 (10355-1-AP, Proteintech; 1:1000 dilution); GAPDH (10494-1-AP, Proteintech; 1:10000 dilution).

Techniques: Binding Assay, ChIP-sequencing, Transfection, Western Blot, Quantitative RT-PCR, Infection, ChIP-qPCR

Journal: Cell Death & Disease

Article Title: The RBPJ/DAPK3/UBE3A signaling axis induces PBRM1 degradation to modulate the sensitivity of renal cell carcinoma to CDK4/6 inhibitors

doi: 10.1038/s41419-022-04760-6

Figure Lengend Snippet: DAPK3 competed with PKA to bind with UBE3A and enhance the PBRM1 degradation in renal cancer cells. PBPJ transcriptionally regulated DAPK3 expression and then promoted UBE3A-mediated degradation of PBRM1. Then, PBRM1 increased the p21 expression and sensitized renal cancer cells to CDK4/6 inhibitors. In combination with RBPJ inhibitors, CDK4/6 inhibitors synergistically enhanced renal cancer cells.

Article Snippet: The antibodies used as follows: UBE3A (10344-1-AP, Proteintech; 1:1000 dilution), PBRM1 (12563-1-AP, Proteintech; 1:500 dilution), DAPK3 (2928, Cell signaling technology, 1:1000 dilution), RBPJ (14613-1-AP, Proteintech; 1:1000 dilution); P21 (10355-1-AP, Proteintech; 1:1000 dilution); GAPDH (10494-1-AP, Proteintech; 1:10000 dilution).

Techniques: Expressing

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, Virus, Two Tailed Test, Generated, Expressing, shRNA, Quantitative RT-PCR

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: Genome-wide analysis of chromatin accessibility and gene expression in iNSCs and NSCs. a Heatmaps of ATAC-seq signals for miNSC10 and SCR029 cells within an 8 kb window centered around the peak summit. b Gene ontology (GO) enrichment analysis of the genes associated with miNSC10 ATAC-seq peaks. They were analyzed for GO term enrichment by BiNGO and the result was visualized on a network of gene sets (nodes) connected by their similarity (edges). Node size represents the gene-set size and edge thickness represents the degree of overlap between two gene sets. Depicted are two prominent groups of enriched gene sets. c The 10 top-ranked TF-binding motifs for both miNSC10 and SCR029 cells identified by de novo motif search in a 300-bp window centered at the peak summit. d – g Genome browser view of ATAC-seq and RNA-seq signals at the Olig2 , Sox2 , Pou3f2 , and Pax6 loci in MEF, miNSC10, and SCR029 cells. The y axis represents the number of normalized reads. h Schematic showing the process by which Ptf1a directly reprograms somatic cells into tripotent iNSCs and the associated molecular changes. Ptf1a must form DNA-binding complex with Rbpj to directly or indirectly activate expression of several families (Sox, bHLH, homeobox, POU, Nfi, and Rfx) of transcription factor genes involved in NSC self-renewal and maintenance

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: Genome Wide, Gene Expression, Binding Assay, RNA Sequencing, Expressing

Journal: Cell reports

Article Title: Autophagy Controls CSL/RBPJκ Stability through a p62/SQSTM1-Dependent Mechanism

doi: 10.1016/j.celrep.2018.08.043

Figure Lengend Snippet: (A and B) Proximity ligation assays (PLAs) for in situ detection of CSL and p62 association in HDFs. Assays were carried out in the presence or absence of primary antibodies (A) or with HDFs plus/minus siRNA-mediated CSL gene silencing (B) as controls of specificity. Confocal microscopy was used to examine red fluorescence PLA puncta with concomitant DAPI staining of nuclei (blue). The white frames indicate the enlarged areas to better show PLA red puncta. Shown are representative images and quantification of the average number of puncta per cell, counting at least 30 cells in four fields per conditions. p ≤ 0.05, two-tailed unpaired t test. Scale bars, 10 and 1 μm for enlargements. Additional PLA images of p62 and CSL interaction are provided in . (C) Endogenous p62 and CSL proteins interact. Immunoprecipitates from HDFs (strain SAN4) with anti-CSL antibodies and non-immune immunoglobulin Gs (IgGs) were analyzed, together with total inputs by sequential immunoblotting with antip62 and CSL antibodies. IgG light chains (LCs) are indicated. A similar immunoprecipitation in another HDF strain (SAN3) is shown in . (D) p62 and CSL proteins directly interact. Recombinant CSL and p62 proteins were admixed and incubated for 2 hr before immunoprecipitation with either anti-CSL or anti-p62 antibodies or nonimmune IgGs, followed by immunoblot analysis with anti-p62 antibodies (green) and anti-CSL antibodies (red). IgG heavy chains (HCs) are indicated. (E) p62 interaction with CSL requires p62 C-terminal region. Immunoblot analysis showed HEK293 transfected for 48 hr with expression vectors for CSL-FLAG, GFP-p62, and a combination of CSLFLAG and GFP-p62 wild-type (WT) or GFP-p62 constructs with deletions of the N-terminal PB1 region, LIR, and C-terminal UBA region (as indicated in the scheme) . Total lysates were immunoprecipitated with anti-FLAG antibodies, and blots were probed sequentially with anti-GFP and anti-FLAG antibodies. The input totals were analyzed by parallel immunoblotting as control for the level of expression.

Article Snippet: Recombinant human CSL , Origene , Cat# TP760429.

Techniques: Ligation, In Situ, Confocal Microscopy, Fluorescence, Staining, Cell Counting, Two Tailed Test, Western Blot, Immunoprecipitation, Recombinant, Incubation, Transfection, Expressing, Construct, Control

Journal: Cell reports

Article Title: Autophagy Controls CSL/RBPJκ Stability through a p62/SQSTM1-Dependent Mechanism

doi: 10.1016/j.celrep.2018.08.043

Figure Lengend Snippet:

Article Snippet: Recombinant human CSL , Origene , Cat# TP760429.

Techniques: Recombinant, Proximity Ligation Assay, Control, shRNA, Software

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, Virus, Two Tailed Test, Generated, Expressing, shRNA, Quantitative RT-PCR

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: Genome-wide analysis of chromatin accessibility and gene expression in iNSCs and NSCs. a Heatmaps of ATAC-seq signals for miNSC10 and SCR029 cells within an 8 kb window centered around the peak summit. b Gene ontology (GO) enrichment analysis of the genes associated with miNSC10 ATAC-seq peaks. They were analyzed for GO term enrichment by BiNGO and the result was visualized on a network of gene sets (nodes) connected by their similarity (edges). Node size represents the gene-set size and edge thickness represents the degree of overlap between two gene sets. Depicted are two prominent groups of enriched gene sets. c The 10 top-ranked TF-binding motifs for both miNSC10 and SCR029 cells identified by de novo motif search in a 300-bp window centered at the peak summit. d – g Genome browser view of ATAC-seq and RNA-seq signals at the Olig2 , Sox2 , Pou3f2 , and Pax6 loci in MEF, miNSC10, and SCR029 cells. The y axis represents the number of normalized reads. h Schematic showing the process by which Ptf1a directly reprograms somatic cells into tripotent iNSCs and the associated molecular changes. Ptf1a must form DNA-binding complex with Rbpj to directly or indirectly activate expression of several families (Sox, bHLH, homeobox, POU, Nfi, and Rfx) of transcription factor genes involved in NSC self-renewal and maintenance

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: Genome Wide, Gene Expression, Binding Assay, RNA Sequencing, Expressing