Journal: The Journal of Biological Chemistry

Article Title: The N-acetylglucosaminyltransferase Radical fringe contributes to defects in JAG1-dependent turnover and signaling of NOTCH3 CADASIL mutants

doi: 10.1016/j.jbc.2024.107787

Figure Lengend Snippet: RFNG does not alter the steady-state levels of the NOTCH3 WT or C185R protein. A , representative Western blot of lysates from cultured N3WT/C185R-RF-HeLa cells with and without doxycycline-inducible expression of RFNG. NOTCH3 and RFNG were detected by antibodies against human NOTCH3 extracellular domain (ECD) and human RFNG. TUBULIN was blotted with an antibody against human/mouse α-tubulin as a loading control. The white and black arrowheads indicate full-length NOTCH3 and the ECD, respectively. B , representative Western blot of lysates from cultured N3WT/C185R-RF-HeLa cells treated with or without 0, 0.1, 0.5, or 1 μg/ml doxycycline. RFNG was detected by an antibody against HA, which tagged RFNG. The black arrowheads indicate N3ECDs. C , relative amounts of N3ECD WT and C185R with or without RFNG expression, as determined by Western blotting (B) (independent biological replicates N = 3). N3ECD band intensities were normalized to those observed without doxycycline. D , representative Western blot of cell lysates collected from cultured N3WT-RF-HeLa and N3C185R-RF-HeLa cells with or without RFNG expression and treated with puromycin for 0 or 16 h. The black arrowheads indicate N3ECDs. E , relative band intensities of N3ECD at 16 h normalized to those observed at 0 h, as determined by Western blotting in (D) (independent biological replicates N = 3). Data information: In C and E , data are presented as mean ± SD. n.s., not significant (C: Tukey’s post hoc test following one-way ANOVA; E: Tukey’s post hoc test following two-way ANOVA). HA, hemagglutinin; N3ECD, NOTCH3 extracellular domain; RFNG, Radical fringe.

Article Snippet: The cells were then cocultured with JAG1-3T3 cells (2.0 × 10 5 cells/well) and treated with 200 μM leupeptin hemisulfate for 8 h. The cells were fixed with 4% PFA for 10 min, blocked with 1% Block Ace (Pharma Biomedical) for 30 min, and incubated with mouse anti-human N3ECD (Abnova, 1G5; 1:200) in 0.4% Block Ace for 60 min. After permeabilization with 0.2% Triton X-100 in PBS for 5 min, the cells were reblocked with 1% Block Ace and incubated with sheep anti-human N3ECD (R&D Systems, BAF1559; 1:400) in 0.4% Block Ace and 0.1% Triton X-100 for 60 min.

Techniques: Western Blot, Cell Culture, Expressing, Control

Journal: The Journal of Biological Chemistry

Article Title: The N-acetylglucosaminyltransferase Radical fringe contributes to defects in JAG1-dependent turnover and signaling of NOTCH3 CADASIL mutants

doi: 10.1016/j.jbc.2024.107787

Figure Lengend Snippet: RFNG enhances JAG1-induced degradation of NOTCH3 WT but not that of R141C and C185R. A , images of immunocytochemistry with an N3ECD antibody after coculturing N3WT/C185R-RF-HeLa with MIG-3T3 or JAG1-3T3 cells. N3WT/C185R-RF-HeLa was cultured with and without dox for 1 day before being cocultured with MIG-3T3 or JAG1-3T3 for 1 day. The left panel shows GFP expressed in MIG-3T3 and JAG1-3T3 ( green ). The middle panel shows N3ECD ( magenta ). The right panel shows an overlay. Arrows indicate N3ECD transendocytosed into JAG1-3T3. The scale bar represents 10μm. B , proportion of JAG1-3T3 and MIG-3T3 positive with NOTCH3 WT and C185R (independent biological replicates, N = 4). C , representative images of immunocytochemistry with an N3ECD antibody after coculturing N3WT/R141C-RF-HeLa with JAG1-3T3 cells. N3WT/R141C-RF-HeLa was cultured with and without dox for 1 day before being cocultured with MIG-3T3 or JAG1-3T3 for 1 day. The scale bar represents 10μm. D , proportion of JAG1-3T3 and MIG-3T3 positive with NOTCH3 WT and R141C (independent biological replicates, N = 5). Data information: In B and D , data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, n.s., not significant (Tukey’s post hoc test following three-way ANOVA). JAG1, Jagged 1; N3ECD, NOTCH3 extracellular domain; RFNG, Radical fringe.

Article Snippet: The cells were then cocultured with JAG1-3T3 cells (2.0 × 10 5 cells/well) and treated with 200 μM leupeptin hemisulfate for 8 h. The cells were fixed with 4% PFA for 10 min, blocked with 1% Block Ace (Pharma Biomedical) for 30 min, and incubated with mouse anti-human N3ECD (Abnova, 1G5; 1:200) in 0.4% Block Ace for 60 min. After permeabilization with 0.2% Triton X-100 in PBS for 5 min, the cells were reblocked with 1% Block Ace and incubated with sheep anti-human N3ECD (R&D Systems, BAF1559; 1:400) in 0.4% Block Ace and 0.1% Triton X-100 for 60 min.

Techniques: Immunocytochemistry, Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: The N-acetylglucosaminyltransferase Radical fringe contributes to defects in JAG1-dependent turnover and signaling of NOTCH3 CADASIL mutants

doi: 10.1016/j.jbc.2024.107787

Figure Lengend Snippet: RFNG does not affect JAG1-mediated transendocytosis of NOTCH3 WT and C185R. A , schematic representation of the immunocytochemistry workflow used to separately detect NOTCH3 present in the cell membrane and in JAG1-3T3 cells. After N3WT/C185R-RF-HeLa cells transfected with NOTCH2 siRNA were cocultured with JAG1-3T3 cells, the fixed cells were stained with two different anti-NOTCH3ECD (N3ECD) antibodies (clones 1G5 and BAF1559) before and after cell permeabilization, respectively. The scale bars indicate 20 μm. B , representative confocal images of cocultured N3WT/C185R-RF-HeLa and JAG1-3T3 cells, as depicted in (A). The white arrowheads indicate NOTCH3 particles detected by antibodies against N3ECD (clones 1G5 and BAF1559), indicating the presence of NOTCH3 on the cell membrane. The scale bars indicate 20 μm. C , the ratio of NOTCH3 present on the cell membrane against the total NOTCH3 in JAG1-3T3 cells. Box plots: 10 to 90 percentile. (independent biological replicates: N = 3; total cell numbers, WT RFNG-: n = 86, WT RFNG+: n = 86, C185R RFNG-: n = 93, C185R RFNG+: n = 81). ∗∗p < 0.01, ∗∗∗∗ p < 0.0001 (Tukey’s post hoc tests following two-way ANOVA). JAG1, Jagged 1; N3ECD, NOTCH3 extracellular domain; RFNG, Radical fringe.

Article Snippet: The cells were then cocultured with JAG1-3T3 cells (2.0 × 10 5 cells/well) and treated with 200 μM leupeptin hemisulfate for 8 h. The cells were fixed with 4% PFA for 10 min, blocked with 1% Block Ace (Pharma Biomedical) for 30 min, and incubated with mouse anti-human N3ECD (Abnova, 1G5; 1:200) in 0.4% Block Ace for 60 min. After permeabilization with 0.2% Triton X-100 in PBS for 5 min, the cells were reblocked with 1% Block Ace and incubated with sheep anti-human N3ECD (R&D Systems, BAF1559; 1:400) in 0.4% Block Ace and 0.1% Triton X-100 for 60 min.

Techniques: Immunocytochemistry, Membrane, Transfection, Staining, Clone Assay

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: Targeting the Notch-Furin axis with 2-hydroxyoleic acid: a key mechanism in glioblastoma therapy

doi: 10.1007/s13402-024-00995-x

Figure Lengend Snippet: Sequence of primers used in the PCR assays

Article Snippet: The cells were then incubated with ammonium chloride (100 mM: Sigma) at room temperature for 10 min and they were then stained overnight with primary antibodies against Notch3 (Santa Cruz Biotechnology, Heidelberg, Germany), Notch2 (BioLegend, Barcelona, Spain), Hes1 (Santa Cruz Biotechnology, Heidelberg, Germany) and Giantin (Biolegend, Barcelona, Spain), all diluted 1:100.

Techniques: Sequencing

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: Targeting the Notch-Furin axis with 2-hydroxyoleic acid: a key mechanism in glioblastoma therapy

doi: 10.1007/s13402-024-00995-x

Figure Lengend Snippet: 2OHOA inhibits Notch signaling pathway. a Representative immunoblots of Notch2/3 FL (full length), Notch2/3 TM (transmembrane domain) and NICD2/3 (Notch intracellular domain) proteins after exposure of U-87 MG cells to 2OHOA (200 µM) for 6, 12, 24 and 48 h (four independent experiments with two replicates each). b The Hes1 protein levels after exposure to 2OHOA (200 µM) for 48 h as an indicator of its pharmacological effect in U-87 MG cells. c The mRNA expression of JAGGED1 , NOTCH1 , NOTCH2 , NOTCH3 , HES1 and CD3 genes in U-87 MG treated for 48 h with 2OHOA (200 µM) or the vehicle alone. The values are presented as the mean ± SEM of at least four independent experiments analyzed in triplicate. d JAGGED1 mRNA levels in U-87 MG cells exposed to 2OHOA (200 µM) or the vehicle alone for 72 h. The results are from three independent experiments with three replicates each and they are expressed as the % of the control ± SEM. e Cell localization of Notch3 (left panels) and Hes1 (right panels) in U-87 MG control cells (row 1, 2) or those exposed to 2OHOA (400 µM) for 48 h (row 3, 4). Images were taken at 40× magnification. Scale bar: 20 μm (row 1, 3), 5 μm (row 2,4). f Nuclear fluorescence intensity of Notch3 (upper graph) or Hes1 (lower graph) in two independent experiments. The statistical analysis was performed with a t -test with Welch’s correction relative to the untreated cells: *** p < 0.001

Article Snippet: The cells were then incubated with ammonium chloride (100 mM: Sigma) at room temperature for 10 min and they were then stained overnight with primary antibodies against Notch3 (Santa Cruz Biotechnology, Heidelberg, Germany), Notch2 (BioLegend, Barcelona, Spain), Hes1 (Santa Cruz Biotechnology, Heidelberg, Germany) and Giantin (Biolegend, Barcelona, Spain), all diluted 1:100.

Techniques: Western Blot, Expressing, Control, Fluorescence

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: Targeting the Notch-Furin axis with 2-hydroxyoleic acid: a key mechanism in glioblastoma therapy

doi: 10.1007/s13402-024-00995-x

Figure Lengend Snippet: Graphical illustration describing the 2OHOA mechanism of action to inhibit Notch2 and Notch3 signaling. Model for activated Notch2/3 signaling (left panel). Proposal model for 2OHOA mechanism of action (right panel) where its interaction with the furin enzyme prevents Notch2 processing in Golgi. Instead, Notch3 is transcriptionally repressed probably by indirect 2OHOA mechanism (indicated as dashed line). In addition, HES1 overexpression partially inhibits the 2OHOA effect on viability (narrower dashed line). Continuous lines indicate activation while transparent continuous lines represent lower activation of the pathway

Article Snippet: The cells were then incubated with ammonium chloride (100 mM: Sigma) at room temperature for 10 min and they were then stained overnight with primary antibodies against Notch3 (Santa Cruz Biotechnology, Heidelberg, Germany), Notch2 (BioLegend, Barcelona, Spain), Hes1 (Santa Cruz Biotechnology, Heidelberg, Germany) and Giantin (Biolegend, Barcelona, Spain), all diluted 1:100.

Techniques: Over Expression, Activation Assay

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: Targeting the Notch-Furin axis with 2-hydroxyoleic acid: a key mechanism in glioblastoma therapy

doi: 10.1007/s13402-024-00995-x

Figure Lengend Snippet: Sequence of primers used in the PCR assays

Article Snippet: The nitrocellulose membranes were probed overnight at 4°C with the primary antibodies against Notch2 Intracellular domain (Hybridoma Bank, Iowa City, Iowa), Notch3 (Santa Cruz Biotechnology, Heidelberg, Germany) and Hes1 (Santa Cruz Biotechnology, Heidelberg, Germany) diluted 1:1,000.

Techniques: Sequencing

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: Targeting the Notch-Furin axis with 2-hydroxyoleic acid: a key mechanism in glioblastoma therapy

doi: 10.1007/s13402-024-00995-x

Figure Lengend Snippet: 2OHOA inhibits Notch signaling pathway. a Representative immunoblots of Notch2/3 FL (full length), Notch2/3 TM (transmembrane domain) and NICD2/3 (Notch intracellular domain) proteins after exposure of U-87 MG cells to 2OHOA (200 µM) for 6, 12, 24 and 48 h (four independent experiments with two replicates each). b The Hes1 protein levels after exposure to 2OHOA (200 µM) for 48 h as an indicator of its pharmacological effect in U-87 MG cells. c The mRNA expression of JAGGED1 , NOTCH1 , NOTCH2 , NOTCH3 , HES1 and CD3 genes in U-87 MG treated for 48 h with 2OHOA (200 µM) or the vehicle alone. The values are presented as the mean ± SEM of at least four independent experiments analyzed in triplicate. d JAGGED1 mRNA levels in U-87 MG cells exposed to 2OHOA (200 µM) or the vehicle alone for 72 h. The results are from three independent experiments with three replicates each and they are expressed as the % of the control ± SEM. e Cell localization of Notch3 (left panels) and Hes1 (right panels) in U-87 MG control cells (row 1, 2) or those exposed to 2OHOA (400 µM) for 48 h (row 3, 4). Images were taken at 40× magnification. Scale bar: 20 μm (row 1, 3), 5 μm (row 2,4). f Nuclear fluorescence intensity of Notch3 (upper graph) or Hes1 (lower graph) in two independent experiments. The statistical analysis was performed with a t -test with Welch’s correction relative to the untreated cells: *** p < 0.001

Article Snippet: The nitrocellulose membranes were probed overnight at 4°C with the primary antibodies against Notch2 Intracellular domain (Hybridoma Bank, Iowa City, Iowa), Notch3 (Santa Cruz Biotechnology, Heidelberg, Germany) and Hes1 (Santa Cruz Biotechnology, Heidelberg, Germany) diluted 1:1,000.

Techniques: Western Blot, Expressing, Control, Fluorescence

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: Targeting the Notch-Furin axis with 2-hydroxyoleic acid: a key mechanism in glioblastoma therapy

doi: 10.1007/s13402-024-00995-x

Figure Lengend Snippet: Graphical illustration describing the 2OHOA mechanism of action to inhibit Notch2 and Notch3 signaling. Model for activated Notch2/3 signaling (left panel). Proposal model for 2OHOA mechanism of action (right panel) where its interaction with the furin enzyme prevents Notch2 processing in Golgi. Instead, Notch3 is transcriptionally repressed probably by indirect 2OHOA mechanism (indicated as dashed line). In addition, HES1 overexpression partially inhibits the 2OHOA effect on viability (narrower dashed line). Continuous lines indicate activation while transparent continuous lines represent lower activation of the pathway

Article Snippet: The nitrocellulose membranes were probed overnight at 4°C with the primary antibodies against Notch2 Intracellular domain (Hybridoma Bank, Iowa City, Iowa), Notch3 (Santa Cruz Biotechnology, Heidelberg, Germany) and Hes1 (Santa Cruz Biotechnology, Heidelberg, Germany) diluted 1:1,000.

Techniques: Over Expression, Activation Assay