adenovirus treated ikk2ca  (OriGene)

Journal: bioRxiv

Article Title: Rbpj deletion in hepatic progenitor cells attenuates endothelial responses in a mouse model of cholestatic liver disease

doi: 10.1101/2024.04.13.589277

Figure Lengend Snippet: (A) qPCR analysis was performed using HPCs transduced with lentiviral vectors expressing two different shRNAs against Rbpj (shRNA-1 and shRNA-2) and a non-silencing control and then subjected to puromycin selection. Data are expressed as mean ± standard deviation; n = 4 independent experiments. (B) Immunofluorescence analysis. Yellow arrowheads, YFP+OPN+VEGFA+ HPCs. White arrows, YFP-OPN+VEGFA+ cholangiocytes. Scale bar, 50 μm. (C) Quantification of VEGFA+ areas. Data are expressed as mean ± standard deviation; n = 11 mice per group. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns, not significant. Abbreviations: CKO, conditional knockout; HPC, hepatic progenitor cell; OPN, osteopontin; qPCR, quantitative polymerase chain reaction; VEGFA, vascular endothelial growth factor A; WT, wild-type; YFP, yellow fluorescent protein.

Article Snippet: [ , ] HPCs were treated with lentiviral vectors expressing a non-silencing control and each of the 2 different shRNAs targeting the mouse Rbpj gene (VectorBuilder).

Techniques: Transduction, Expressing, shRNA, Selection, Standard Deviation, Immunofluorescence, Knock-Out, Real-time Polymerase Chain Reaction

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: PLoS ONE

Article Title: RBPJ Is a Novel Target for Rhabdomyosarcoma Therapy

doi: 10.1371/journal.pone.0039268

Figure Lengend Snippet: The expression of RBPJ mRNA in RD cells was assessed by real-time PCR. The Ct values of all RMS cell lines were normalized to those of ACTB . The values of the RMS cell lines were compared with HSkMc cell, which is defined as a relative expression of 1.0. B, RBPJ protein levels in RD cells transfected with control and RBPJ siRNA were examined by western blotting analysis (top). RBPJ and HES1 mRNA in RD cells transfected with control and RBPJ siRNA were assessed by real-time PCR analysis. Ct values of RBPJ and HES1 were normalized to ACTB . The values of the cells transfected with RBPJ siRNA were compared to those the RD cells transfected with control siRNA, which is defined as a relative expression of 1.0 (bottom). C, Anchorage-independent growth of RD cells transfected with control and RBPJ siRNA were evaluated by colony formation assay. After 3 weeks, each of the colonies were counted and photographed. Scale bar is 200 µM. Columns, mean values of 3 independent experiments; bar, SD. * p <0.05, ** p <0.01.

Article Snippet: Control or RBPJ shRNA (KH06319P) were purchased from SuperArray Biosciences (MD, USA). pCMV6-Entry Vector (PS100001) and RBPJ expression vector (RC204791) were purchased from Origene (Maryland, USA).

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

Journal: PLoS ONE

Article Title: RBPJ Is a Novel Target for Rhabdomyosarcoma Therapy

doi: 10.1371/journal.pone.0039268

Figure Lengend Snippet: After transfection of control shRNA or RBPJ shRNA, 1×10 6 RD cells were subcutaneously inoculated in nude mice (n = 7). Tumor size was calculated weekly by using the formula LW 2 /2 (with L and W representing the length and width of tumors). B , Survival rate of the mice injected with control shRNA- or RBPJ shRNA-transfected RD cells was assessed by Kaplan–Meier analysis. C , The number of Ki67-positive cells in control shRNA- or RBPJ shRNA-transfected xenograft were accessed by immnohistochemistry. Scale bar is 100 µm. Expression of Cell cycle-related genes ( CyclinD , CyclinE , E2F1 , SKP2 , p21 ) were assessed by real-time PCR in control shRNA- or RBPJ shRNA-transfected xenograft. The Ct values of xenograft samples were normalized to those of ACTB . The values of the RBPJ shRNA-transfected xenograft was compared with those of the control shRNA sample, which is defined as a relative expression of 1.0. Columns, mean values of 3 independent experiments. Bar, SD. * p <0.05, ** p <0.01.

Article Snippet: Control or RBPJ shRNA (KH06319P) were purchased from SuperArray Biosciences (MD, USA). pCMV6-Entry Vector (PS100001) and RBPJ expression vector (RC204791) were purchased from Origene (Maryland, USA).

Techniques: Transfection, shRNA, Injection, Expressing, Real-time Polymerase Chain Reaction

Journal: Cancer Science

Article Title: RBPJ contributes to the malignancy of glioblastoma and induction of proneural‐mesenchymal transition via IL‐6‐STAT3 pathway

doi: 10.1111/cas.14642

Figure Lengend Snippet: Effects of RBPJ knockdown on the proliferation and invasiveness of glioblastoma (GBM) cells. A, Expression of RBPJ in 2 GBM cell lines (U251 and T98), as evaluated by western blotting. HEK293 cells served as a positive control for RBPJ expression. B, Decreased RBPJ protein level in cells transduced with both RBPJ‐targeting small hairpin RNA (shRNA)1 and 2 compared with the control in GBM cells. C, Proliferation assay using Alamar blue. Both shRBPJ1 and 2 suppressed proliferation ability of GBM cells. Data are presented as the mean ± standard deviation (SD) (n = 6). * P < .05; ** P < .01, *** P < .001. D, Invasiveness of RBPJ‐silenced and control GBM cells, as assessed by transwell assay. Invasiveness of RBPJ‐silenced GBM cells was reduced compared with that of control cells. The bars represent mean cell counts from at least 6 high‐powered fields (HPF). * P < .05; ** P < .01, *** P < .001

Article Snippet: Cells (20 × 10 4 cells) were seeded into 6‐well plates and exposed to 1 of the 2 RBPJ‐targeting shRNA lentiviral particles (shRBPJ1, sc270318‐v; Santa Cruz Biotechnology; shRBPJ2, Sigma‐Aldrich) or non‐targeting control (sc108080; Santa Cruz Biotechnology) shRNA lentiviral particles.

Techniques: Expressing, Western Blot, Positive Control, Transduction, shRNA, Proliferation Assay, Standard Deviation, Transwell Assay

Journal: Cancer Science

Article Title: RBPJ contributes to the malignancy of glioblastoma and induction of proneural‐mesenchymal transition via IL‐6‐STAT3 pathway

doi: 10.1111/cas.14642

Figure Lengend Snippet: RBPJ expression in glioblastoma stem‐like cells (GSCs) and the effect of RBPJ knockdown on the proliferation and self‐renewal of GSCs. A, RBPJ expression in GSC lines, as assessed by western blotting. HEK293 cells served as a positive control for RBPJ expression. B, RBPJ expression in GSCs and FBS‐differentiated GBM cells derived from the same patients. RBPJ protein expression of all GSCs we used were significantly higher compared with FBS‐differentiated GBM cells. Stemness and differentiation were confirmed by positive CD133 and glial fibrillary acidic protein (GFAP) staining, respectively. C, RBPJ protein levels in cells transduced with control and RBPJ‐targeting shRNAs, as examined by western blotting. RBPJ protein level was decreased in cells transduced with both shRBPJ1 and shRBPJ2 compared with control for all GSCs. D, Proliferation assay using Alamar blue. RBPJ suppression inhibited the proliferation of all GSCs. Data are presented as means ± SD (n = 4). * P < .05; ** P < .01, *** P < .001. E, Effects of RBPJ knockdown on GSC tumorsphere formation. Numbers of tumor spheres formed by both shRBPJ1 and shRBPJ2 cells were decreased compared with shCONT cells. Data are presented as means ± SD (n = 3). * P < .05; ** P < .01, *** P < .001

Article Snippet: Cells (20 × 10 4 cells) were seeded into 6‐well plates and exposed to 1 of the 2 RBPJ‐targeting shRNA lentiviral particles (shRBPJ1, sc270318‐v; Santa Cruz Biotechnology; shRBPJ2, Sigma‐Aldrich) or non‐targeting control (sc108080; Santa Cruz Biotechnology) shRNA lentiviral particles.

Techniques: Expressing, Western Blot, Positive Control, Derivative Assay, Staining, Transduction, Proliferation Assay

Journal: Cancer Science

Article Title: RBPJ contributes to the malignancy of glioblastoma and induction of proneural‐mesenchymal transition via IL‐6‐STAT3 pathway

doi: 10.1111/cas.14642

Figure Lengend Snippet: RBPJ regulated IL‐6 and phosphorylated STAT3. A, Protein levels of IL‐6 and p‐STAT3 (Y705) after RBPJ knockdown. Protein levels of both IL‐6 and p‐STAT3 (Y705) were decreased in glioblastoma (GBM) cell lines and GBM stem‐like cells (GSCs) transduced with both shRBPJ1 and shRBPJ2 compared with control cells. B, The protein level of p‐STAT3 (Y705) in cells treated with a neutralizing antibody targeting IL‐6. p‐STAT3 (Y705) was decreased with the administration of the neutralizing IL‐6 antibody in all GBM cell lines and GSCs

Article Snippet: Cells (20 × 10 4 cells) were seeded into 6‐well plates and exposed to 1 of the 2 RBPJ‐targeting shRNA lentiviral particles (shRBPJ1, sc270318‐v; Santa Cruz Biotechnology; shRBPJ2, Sigma‐Aldrich) or non‐targeting control (sc108080; Santa Cruz Biotechnology) shRNA lentiviral particles.

Techniques: Transduction

Journal: Cancer Science

Article Title: RBPJ contributes to the malignancy of glioblastoma and induction of proneural‐mesenchymal transition via IL‐6‐STAT3 pathway

doi: 10.1111/cas.14642

Figure Lengend Snippet: RBPJ knockdown in glioblastoma stem‐like cells (GSCs) suppressed tumor growth and increased survival of animals bearing intracranial GBM. A, Hematoxylin and eosin staining, immunofluorescence staining of RBPJ and immunohistochemistry of Ki‐67 for mouse brain tissue implanted with KGS10‐shRBPJ1 or KGS10‐shCONT. The tumor area of KGS10‐shRBPJ1 was clearly smaller than that of the control. Scale bars, 200 μm. B, The Kaplan‐Meier curve of KGS10‐shRBPJ1 group and shCONT group. The survival time of KGS10‐shRBPJ1 group was significantly longer than that of KGS10‐shCONT. C, Immunohistochemical staining showing the expression of CD44, YKL‐40, OLIG2 and PDGFRα. Expression of YKL‐40 was decreased, while that of OLIG2 was increased in tumors formed by KGS10‐shRBPJ1 cells. CD44 was not originally expressed in KGS10 cells and the levels of PDGFRα were not significantly changed. Scale bars, 100 μm

Article Snippet: Cells (20 × 10 4 cells) were seeded into 6‐well plates and exposed to 1 of the 2 RBPJ‐targeting shRNA lentiviral particles (shRBPJ1, sc270318‐v; Santa Cruz Biotechnology; shRBPJ2, Sigma‐Aldrich) or non‐targeting control (sc108080; Santa Cruz Biotechnology) shRNA lentiviral particles.

Techniques: Staining, Immunofluorescence, Immunohistochemistry, Immunohistochemical staining, Expressing

Journal: Communications Biology

Article Title: Reciprocal H3.3 gene editing identifies K27M and G34R mechanisms in pediatric glioma including NOTCH signaling

doi: 10.1038/s42003-020-1076-0

Figure Lengend Snippet: a Dose–response curves of DAPT treatment on our cell panel. Curves were fitted using the “log(inhibitor) versus response–variable slope” model and IC50s were calculated using the GraphPad Prism software. Arrows denote the direction of IC50 shift upon gene editing where applicable. b Dose–response curves of Panobinostat treatment on our cell panel. For a , b , p values comparing LogIC50s were calculated by unpaired t test using GraphPad QuickCalcs with n = 3 biologically independent samples. c Western blot of ASCL1 knockdown in subgroups of our panel of cells using siRNA for ASCL1, with quantification of ASCL1 levels exhibiting strong knockdown in XIII and XVII lines. siRNA targeting noncoding RNA from the same vendor as our targeting siRNA (Thermo) was used as control. d Cell viability measured using CellTiterGlo assay (Promega) of cells under siRNA knockdown of ASCL1 plotted as dot plot with mean ± sd; n = 4 biologically independent samples. e Western blot of ASCL1 levels with transient transfection of empty vector or pCS2-HA-ASCL1 plasmid leading to overexpression of ASCL1 in XIII-WT and XVII-WT cells. f Cell viability of XIII-WT and XVII-WT cells with overexpression of ASCL1, mean ± sd; n = 3 biologically independent samples. g Western blot of RBPJ knockdown in our panel of cell lines. RBPJ knockdown was the strongest in XIII and XVII cells, which exhibit higher RBPJ expression than the H3.3WT-reverted cells. h Cell viability of glioma cell lines under RBPJ knockdown, mean ± sd; n = 3 biologically independent samples. i Western blot of RBPJ levels across cell panel under RIN1 (RBPJ inhibitor 1) treatment (2 μM, 72 h). RIN1 does not significantly impact RBPJ protein levels. j Cell viability of glioma cells under RIN1 treatment, mean ± sd; n = 3 biologically independent samples. All p values were calculated by Student’s t test comparison of treated cells versus control or vehicle. * p < 0.05.

Article Snippet: In the case of siRNA transfection, cells were transfected with one of two siRNAs targeting ASCL1 (Ambion #AM16704-41902 or #AM16704-114403) or a combination of both siRNAs or a single siRNA targeting RBPJ (Ambion #AM16708) according to the manufacturer’s protocol using Genejuice transfection reagent (Millipore Sigma).

Techniques: Software, Western Blot, Transfection, Plasmid Preparation, Over Expression, Expressing

Journal: Communications Biology

Article Title: Reciprocal H3.3 gene editing identifies K27M and G34R mechanisms in pediatric glioma including NOTCH signaling

doi: 10.1038/s42003-020-1076-0

Figure Lengend Snippet: a Differential H3K27me3 binding events to super-enhancer (SE) regions are depicted as a cartoon model leading to derepression of downstream pathways and feed-forward overexpression of oncogenic NOTCH signaling and neurogenesis pathways. b NOTCH signaling pathway consists of upstream ASCL1 expression, leading to inter-cell activation and cleavage of NOTCH1 by γ-secretase, with eventual translocation of NICD to the nucleus where it interacts with transcription factors, including RBPJ, thereby inducing transcription activation of genes, such as HES5 . Increased NOTCH signaling has been described as a hallmark of some cancers leading to increased cell cycle transitions. We showed H3.3 mutant glioma cells have elevated expression of NOTCH pathway genes. Transient overexpression (OE) of ASCL1 is sufficient to increase cell viability of H3.3WT-reverted gliomas. siRNA knockdown (KD) of ASCL1 and RBPJ as well as drug inhibition of γ-secretase with DAPT or RBPJ with RIN1 lead to decreased cell viability of H3.3K27M glioma cells in culture. While this model shows ASCL1 OE upstream and ASCL1 KD downstream in the pathway, changes in ASCL1 expression could act in either position to affect cell viability.

Article Snippet: In the case of siRNA transfection, cells were transfected with one of two siRNAs targeting ASCL1 (Ambion #AM16704-41902 or #AM16704-114403) or a combination of both siRNAs or a single siRNA targeting RBPJ (Ambion #AM16708) according to the manufacturer’s protocol using Genejuice transfection reagent (Millipore Sigma).

Techniques: Binding Assay, Over Expression, Expressing, Activation Assay, Translocation Assay, Mutagenesis, Inhibition

Journal: Molecular medicine reports

Article Title: Activation of the Notch‑Nox4‑reactive oxygen species signaling pathway induces cell death in high glucose‑treated human retinal endothelial cells.

doi: 10.3892/mmr.2018.9637

Figure Lengend Snippet: Figure 6. Inhibition of Notch signaling suppresses Nox4 expression in HRECs. Cells were cultured for 24 h in the presence of: Standard glucose as the CTL, HG, or HG and GSI (50 µM). The expression of Nox4 at the (A) mRNA and (B) protein levels was evaluated. Cells were cultured and transfected with siCTL or siRBPj (10 or 20 nM). (C) Protein and (D) mRNA expression levels of RBPj were measured via immunoblotting and reverse transcription‑quantitative polymerase chain reaction, respectively. (E) Cells were cultured for 24 h in presence of HG alone, or HG and 20 nM siRBPj. Nox4 protein expression levels were evaluated. Data are presented as the mean ± standard deviation. n=3. *P<0.05 vs. CTL or siCTL, #P<0.05 vs. HG or HG+siCTL. (F) Schematic of the role of the Notch‑Nox4‑ROS signaling pathway in HG‑induced HRECs injury. Under the conditions of HG, NICD is expressed and is then translocated to the nucleus; NICD induces the upregulation of Nox4 via complex formation of NICD and RBPj. Increased Nox4 upregulates ROS production that is responsible for the induction of cell death. CTL, control; GSI, γ‑secretase inhibitor; HG, high‑glucose; HRECs, human retinal endothelial cells; NICD, Notch1 intracel lular domain; RBPj, recombination signal‑binding protein J; Nox4, NADPH oxidase 4; ROS, reactive oxygen species; siRNA, small interfering RNA; siCTL, control‑siRNA; siRBPj, RBPj‑siRNA.

Article Snippet: To downregulate the expression levels of Nox4 and RBPj, small interfering (si)RNA-Nox4 (siNox4; sc‐41586; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and siRNA-RBPj (sc-38214; Santa Cruz Biotechnology, Inc.) were transfected (final concentrations, 10 and 20 nM) into cells with RNAiMAX (cat. no. 13778‐075; Invitrogen; Thermo Fisher Scientific, Inc.), respectively.

Techniques: Inhibition, Expressing, Cell Culture, Transfection, Western Blot, Polymerase Chain Reaction, Standard Deviation, Control, Small Interfering RNA

Journal: Cell Death Discovery

Article Title: Notch3/VEGF-A axis is involved in TAT-mediated proliferation of pulmonary artery smooth muscle cells: Implications for HIV-associated PAH

doi: 10.1038/s41420-018-0087-9

Figure Lengend Snippet: PASMCs were seeded into 24-well plates with TAT (200 ng/ml) exposure for different time periods (1–24 h). Then cells were collected for total RNA/protein extraction for monitoring the mRNA levels of VEGF-A, B and C and protein levels of VEGF-A. ( a) TAT significantly increased the VEGF-a mRNA levels. (b) TAT exerted no effects on VEGF-B levels. ( c) TAT exerted no effects on VEGF-C levels. (d) TAT significantly increased the VEGF-A protein levels. (e) PASMCs were exposed to Notch ligand Jagged-1 followed with the detection of VEGF-A levels. Jagged-1 significantly increased VEGF-A levels. ( f ) TAT failed to induce the expression of VEGF-A in RBPJ transfected cells. Representative WBs are shown and statistical results are expressed as means ± SEM of four independent experiments and were analyzed using ANOVA (* P < 0.05 vs. control)

Article Snippet: Flag-tagged CSL-VP16 plasmids were obtained from Dr. Aly Karsan (University of British Columbia, Vancouver, Canada) and control Notch3 siRNA (sc-29798), RBPJ siRNA (sc-41446), and scrambled siRNA (sc-37007) were from Santa Cruz Biotechnology.

Techniques: Protein Extraction, Expressing, Transfection, Control

Journal: Cell Death Discovery

Article Title: Notch3/VEGF-A axis is involved in TAT-mediated proliferation of pulmonary artery smooth muscle cells: Implications for HIV-associated PAH

doi: 10.1038/s41420-018-0087-9

Figure Lengend Snippet: PASMCs were pre-treated with DAPT followed by exposure of cells to TAT and assessment of expression of VEGF-A levels. (a and b) DAPT significantly blocked TAT-mediated upregulation VEGF-A levels ( P < 0.05). (c ) The levels of NICD were substantially inhibited in siRNA-Notch3 transfected cells. (d ) SiRNA-Notch3 transfection significantly blocked TAT-mediated upregulation of VEGF-A levels ( P < 0.05). (e) SiRNA-RBPJ transfected cells failed to exhibit TAT-mediated upregulation of VEGF-A levels ( P < 0.05). Representative WBs are shown and statistical results are expressed as means ± SEM of four independent experiments and were analyzed using ANOVA (* P < 0.05 vs. control; # P < 0.05 vs. TAT-treated group)

Article Snippet: Flag-tagged CSL-VP16 plasmids were obtained from Dr. Aly Karsan (University of British Columbia, Vancouver, Canada) and control Notch3 siRNA (sc-29798), RBPJ siRNA (sc-41446), and scrambled siRNA (sc-37007) were from Santa Cruz Biotechnology.

Techniques: Expressing, Transfection, Control