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: 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: The Journal of Clinical Investigation

Article Title: DNA methylation–mediated Rbpjk suppression protects against fracture nonunion caused by systemic inflammation

doi: 10.1172/JCI168558

Figure Lengend Snippet: Chondrogenic pellet and osteogenic differentiation assays were performed within Ikk2ca fl/+ PPCs following Ad-GFP (control) or Ad-Cre ( Ikk2ca ) transfection. ( A ) Alcian blue and alizarin red staining of chondrogenic pellet sections and osteogenic cultures on day 28 and day 21, respectively ( n = 3). ( B ) Real-time qPCR analyses of gene expression for Ikk2 , Sox9 , Col2a1 , Alp , and Spp1 in control or Ikk2ca PPCs ( n = 3). ( C ) ABH/OG staining of fracture callus sections from Ikk2ca fl/+ (control) and Prx1Cre ERT2 ;Ikk2ca fl/+ ( Ikk2ca Prx1 ) mice at 7, 10, and 14 dpf ( n = 5). Scale bars: 100 mm. ( D ) Histomorphometric quantifications of mesenchyme, cartilage, and bone areas based on ABH/OG staining ( n = 5). ( E ) Micro-CT reconstruction of mineralized bony calluses from Ikk2ca fl/+ and Ikk2ca Prx1 mice at 14 dpf ( n = 5). Scale bars: 0.5 mm. ( F ) Bony callus volume and BV/TV measured on micro-CT assessment of Ikk2ca fl/+ and Ikk2ca Prx1 fractures at 14 dpf ( n = 5). Data presented as mean ± SD. * P < 0.05 by 2-tailed Student’s t test.

Article Snippet: After recovery, Lenti- Dnmt3b (MOI = 10; Origene, MR225598L4V) and Lenti-sh Rbpjk (MOI = 10; Origene, TL512813V) were used to infect adenovirus-treated Ikk2ca fl/+ PPCs to overexpress Dnmt3b and knock down Rbpjk in cells, respectively.

Techniques: Control, Transfection, Staining, Gene Expression, Micro-CT

Journal: The Journal of Clinical Investigation

Article Title: DNA methylation–mediated Rbpjk suppression protects against fracture nonunion caused by systemic inflammation

doi: 10.1172/JCI168558

Figure Lengend Snippet: ( A ) Real-time qPCR analyses for Rbpjk gene expression within Ikk2ca fl/+ PPCs following Ad-GFP (control) or Ad-Cre ( Ikk2ca ) transfection ( n = 3). ( B ) Chondrogenic pellet and osteogenic differentiation assays were performed within control and Ikk2ca PPCs in the presence or absence of Lenti-sh Rbpjk ( Rbpjk LOF) viral infection. Alcian blue and alizarin red staining of chondrogenic pellet sections and osteogenic cultures on day 28 and day 21, respectively ( n = 3). ( C ) ABH/OG staining of fracture callus sections from Ikk2ca fl/+ (control), Prx1Cre ERT2 ;Ikk2ca fl/+ ( Ikk2ca Prx1 ), Prx1Cre ERT2 ;Rbpjk fl/fl ( Rbpjk Prx1 ), and Prx1Cre ERT2 ;Ikk2ca fl/+ ;Rbpjk fl/fl ( Ikk2ca;Rbpjk Prx1 ) mice at 7, 10, and 14 dpf ( n = 5). Scale bars: 100 mm. ( D ) Micro-CT reconstruction of mineralized bony calluses from control, Ikk2ca Prx1 , Rbpjk Prx1 , and Ikk2ca;Rbpjk Prx1 mice at 14 dpf ( n = 5). Scale bars: 0.5 mm. Data expressed as mean ± SD. * P < 0.05 determined by 2-tailed Student’s t test for comparisons between 2 groups.

Article Snippet: After recovery, Lenti- Dnmt3b (MOI = 10; Origene, MR225598L4V) and Lenti-sh Rbpjk (MOI = 10; Origene, TL512813V) were used to infect adenovirus-treated Ikk2ca fl/+ PPCs to overexpress Dnmt3b and knock down Rbpjk in cells, respectively.

Techniques: Gene Expression, Control, Transfection, Infection, Staining, Micro-CT

Journal: The Journal of Clinical Investigation

Article Title: DNA methylation–mediated Rbpjk suppression protects against fracture nonunion caused by systemic inflammation

doi: 10.1172/JCI168558

Figure Lengend Snippet: ( A ) Methylation qPCR for the Rbpjk promoter region in Ikk2ca fl/+ PPCs following Ad-GFP (control) or Ad-Cre ( Ikk2ca ) transfection ( n = 3). ( B ) IHC for Dnmt3b on callus sections from Ikk2ca fl/+ (control) and Prx1Cre ERT2 ;Ikk2ca fl/+ ( Ikk2ca Prx1 ) mice at 10 dpf ( n = 5). Scale bars: 100 mm. ( C ) Real-time qPCR analyses of gene expression for Dnmt1 , Dnmt3a , and Dnmt3b in control and Ikk2ca PPCs ( n = 3). ( D ) Real-time qPCR analyses for Rbpjk within control and Ikk2ca PPCs in the presence or absence of Lenti- Dnmt3b ( Dnmt3b GOF) viral infection ( n = 3). ( E ) Schematic representing the selection of C3H10T1/2 cell lines modified by the dCas9-Dnmt3a epigenetic editing system specifically targeting CpG islands of the Rbpjk gene. ( F ) Western blot analyses for Rbpjk in protein extracts from dCas9-Dnmt3a-scramble gRNA (control) and 12 individual dCas9-Dnmt3a-Rbpjk gRNA-engineered C3H10T1/2 cell lines ( n = 3). Lane C: dCas9-Dnmt3a-scramble gRNA. Lanes 1–10: dCas9-Dnmt3a-Rbpjk 1-1-10 gRNAs targeting CpG island 1 of the Rbpjk gene. Lanes 11 and 12: dCas9-Dnmt3a-Rbpjk 2-1-2 gRNAs targeting CpG island 2 of the Rbpjk gene. ( G ) Chondrogenic pellet and osteogenic differentiation assays were performed within dCas9-Dnmt3a-scramble (control) and dCas9-Dnmt3a-Rbpjk 1-9 C3H10T1/2 cell lines in the presence or absence of IL-1β. Alcian blue and alizarin red staining of chondrogenic and osteogenic cultures on day 28 and day 21, respectively ( n = 3). Data expressed as mean ± SD. * P < 0.05 determined by 2-tailed Student’s t test ( A and C ) or by 2-way ANOVA followed by Tukey’s test ( D ).

Article Snippet: After recovery, Lenti- Dnmt3b (MOI = 10; Origene, MR225598L4V) and Lenti-sh Rbpjk (MOI = 10; Origene, TL512813V) were used to infect adenovirus-treated Ikk2ca fl/+ PPCs to overexpress Dnmt3b and knock down Rbpjk in cells, respectively.

Techniques: Methylation, Control, Transfection, Gene Expression, Infection, Selection, Modification, Western Blot, Staining

Journal: The Journal of Clinical Investigation

Article Title: DNA methylation–mediated Rbpjk suppression protects against fracture nonunion caused by systemic inflammation

doi: 10.1172/JCI168558

Figure Lengend Snippet: ( A ) Schematic demonstration of previously established NF-κB/Dnmt3b/Rbpjk axis in regulating fracture repair. Dashed line: Relationship established by in vitro experiments. Solid line: Relationship established in mice. Dashed circle: Role of the gene established in vitro. Solid circle: Role of the gene established in mice. ( B ) Chondrogenic pellet and osteogenic differentiation assays were performed within control and Ikk2ca PPCs in the presence or absence of Lenti- Dnmt3b ( Dnmt3b GOF) viral infection. Alcian blue and alizarin red staining of chondrogenic pellet sections and osteogenic cultures on day 28 and day 21, respectively ( n = 3). ( C ) ABH/OG staining of fracture callus sections from Ikk2ca fl/+ (control), Prx1Cre ERT2 ;Ikk2ca fl/+ ( Ikk2ca Prx1 ), Prx1Cre ERT2 ;Rosa-rtTA fl/+ ;Dnmt3b -Tg ( Dnmt3b-tg Prx1 ), and Prx1Cre ERT2 ;Ikk2ca fl/+ ;Rosa-rtTA fl/+ ;Dnmt3b -Tg ( Ikk2ca;Dnmt3b-tg Prx1 ) mice at 7 and 10 dpf ( n = 5). Scale bars: 100 mm. ( D ) ABH/OG staining of fracture callus sections from Dnmt3b fl/fl (control), Prx1Cre ERT2 ;Dnmt3b fl/fl ( Dnmt3b Prx1 ), Prx1Cre ERT2 ;Rbpjk fl/fl ( Rbpjk Prx1 ), and Prx1Cre ERT2 ; Dnmt3b fl/fl ;Rbpjk fl/fl ( Dnmt3b;Rbpjk Prx1 ) mice at 7 and 10 dpf ( n = 5). Scale bars: 100 mm.

Article Snippet: After recovery, Lenti- Dnmt3b (MOI = 10; Origene, MR225598L4V) and Lenti-sh Rbpjk (MOI = 10; Origene, TL512813V) were used to infect adenovirus-treated Ikk2ca fl/+ PPCs to overexpress Dnmt3b and knock down Rbpjk in cells, respectively.

Techniques: In Vitro, Control, Infection, Staining

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