Journal: The Journal of Biological Chemistry

Article Title: Fringe GlcNAc-transferases differentially extend O -fucose on endogenous NOTCH1 in mouse activated T cells

doi: 10.1016/j.jbc.2022.102064

Figure Lengend Snippet: Endogenous N1 from preT 2017 cells was modified by O -fucose at high stoichiometry, but only the EGF16 O- fucose was extended by Fringes. A , summary of O -fucose modifications identified by mass spectral analysis of endogenous mN1 isolated from preT 2017 cells. Most extended form of the O -fucose glycan detected is shown. Mass spectral data in Fig. S10 and Table S2 . B , extracted ion chromatogram showing relative amounts of O -fucose peptide glycoforms from EGF12 and EGF16. Black , red , and blue lines indicate the unmodified, O -fucose monosaccharide, and both O -fucose and O -glucose monosaccharide glycoforms in the EGF12 panel, respectively. Black , red , blue , green , and magenta lines indicate the unmodified, monosaccharide, disaccharide, trisaccharide, and tetrasaccharide O -fucose glycoforms in the EGF16 panel, respectively. C , cell surface N1 expression measured by flow cytometry in preT 2017 cells or in HEK293T cells overexpressing (O/E) mN1 or empty vector (EV) control. Note that the mouse N1 antibody used here has slight crossreactivity with human N1. D , Western blot analysis of endogenous mN1 in preT 2017 lysate or overexpressed mN1 from HEK293T lysate with anti-N1 ECD antibody. ECD, extracellular domain; EGF, epidermal growth factor-like.

Article Snippet: For cell surface mN1 detection, washed cells were incubated with sheep anti-mN1 antibody (AF5267; R&D Systems, 1:1000 dilution) or sheep nonspecific IgG (R&D Systems, 1:1000 dilution) on ice for 1 h. N1 antibody or nonspecific IgG were detected by antisheep Fc PE conjugate antibody (Thermo, 1/25).

Techniques: Modification, Isolation, Glycoproteomics, Expressing, Flow Cytometry, Plasmid Preparation, Control, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Fringe GlcNAc-transferases differentially extend O -fucose on endogenous NOTCH1 in mouse activated T cells

doi: 10.1016/j.jbc.2022.102064

Figure Lengend Snippet: EGF12 is not modified by Fringe in Fng tKO activated T cells. A , EICs show relative levels of mN1 control peptide lacking an O- fucose site from EGF12 ( black line : 475 QCICMPGYEGVY 486 ) versus mN1 EGF12 peptide with monosaccharide O -fucose modification ( red line : 445 TGPRCEIDVNECISNPCQNDA T CLDQIGEF 474 , O -fucose site bold underlined) from Fng LMR or Fng tKO activated T cells. B , ratio of EGF12 peptide to control peptide from mN1 expressed in HEK293T cells in the absence or presence of LFNG (shown in Fig. S9 ) or mN1 isolated from Fng LMR or Fng tKO activated T cells. NS ≥ 0.05, ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. Mass spectral data for EGF12 and control peptide are shown in Table S4 . Error bars reflect ± SD. EGF, epidermal growth factor-like; EIC, extracted ion chromatogram; tKO, triple knockout.

Article Snippet: For cell surface mN1 detection, washed cells were incubated with sheep anti-mN1 antibody (AF5267; R&D Systems, 1:1000 dilution) or sheep nonspecific IgG (R&D Systems, 1:1000 dilution) on ice for 1 h. N1 antibody or nonspecific IgG were detected by antisheep Fc PE conjugate antibody (Thermo, 1/25).

Techniques: Modification, Control, Isolation, Triple Knockout

Journal: EMBO Molecular Medicine

Article Title: NOTCH1 intracellular domain stabilization by MDM2 plays a major role in NSCLC response to platinum

doi: 10.1038/s44321-025-00354-9

Figure Lengend Snippet: ( A ) 293 T cells were transfected to express NOTCH1-DeltaEx, 6His-ubiquitin (UB-HIS) or empty vector (pcDNA), as indicated, and incubated (+) or not (−) with Carboplatin for 48 h. Histidine pull-down was performed and NICD ubiquitination was analyzed by western blotting ( n = 3). INPUT, total cell extracts. ( B ) Western blotting of the indicated proteins in H358 cells incubated with 100 μM Carboplatin with or without 10 μM KU-55933 (ATM inhibitor) for the indicated time ( n = 3). ( C ) Western blotting of the indicated proteins in H358 cells in which MDM2 was silenced with a siRNA against MDM2 ( siMDM2 ) or not (non-targeting siRNA ; siNT ) ( n = 2). At 6 h post-transfection, 100 μM Carboplatin was added to the cells for 48 h. ( D ) H358 cells transfected with siMDM2 or siNT were incubated with 100 μM Carboplatin for 48 h and with 50 μg/μl of Cycloheximide (CHX) for the indicated time before the end of Carboplatin treatment ( n = 3). Upper panel: schematic representation of the experimental design. ( E ) Graph showing the mean ± SEM of three different experiments performed as described in ( D ) and the statistics between curves was determined using a modified Chi-square-based method. NICD level was normalized to TUBULIN at each time point and then to the level in the untreated sample (lane 0) set to 100%. Hours correspond to Cycloheximide treatment. In the legend, n indicates the number of biological replicates. .

Article Snippet: Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and analyzed using the following antibodies against: NICD (#4147, 1:500), phosphorylated ATM (#13050, 1:1000), ATM (#2873S, 1:1000), phosphorylated ATR (#2853, 1:1000), ATR (#2790S, 1:1000), JAG1 (70109S, 1:1000), JAG2 (2210T, 1:1000), DLL3 (78110S 1:1000), DLL4 (96406S, 1:1000), full length NOTCH1 (3608S, 1:1000), GAPDH (2118S, 1:5000), and H3 (59715S, 1:5000) (all from Cell Signaling Technology, Inc) and also DLL1 (QC16739-41705, Antiva Biosciences, 1:1000), MDM2 (#4B2C1.11 and #4B11 Merck Millipore, 1:2000), TUBULIN (#T9026, Sigma, 1:10,000), polyhistidine (H1029 Sigma, 1:1000), p53 (sc-126 Santa Cruz Technology, 1:1000), TBP (TFIID sc-56794, Santa Cruz Technology, 1:500), HSC70 (sc-7298 Santa Cruz Technology, 1:1000), and HEY1 (#19929 Proteintech 1:1000).

Techniques: Transfection, Ubiquitin Proteomics, Plasmid Preparation, Incubation, Western Blot, Modification

Journal: EMBO Molecular Medicine

Article Title: NOTCH1 intracellular domain stabilization by MDM2 plays a major role in NSCLC response to platinum

doi: 10.1038/s44321-025-00354-9

Figure Lengend Snippet: ( A ) 293 T cells were transfected with NOTCH1-DeltaEx and then co-transfected with empty vector (pcDNA), MDM2 wild-type (WT), MDM2 464 A or MDM2 395 A for 48 h. Then, NICD levels in the chromatin fraction were analyzed by western blotting after cell fractionation ( n = 3). TBP: loading control for the chromatin fraction. ( B ) 293T cells were transfected with NOTCH1-DeltaEx and then co-transfected with 6xtag-His-ubiquitin (UB-HIS), empty vector (pcDNA), MDM2 WT, MDM2 464 A or MDM2 395 A for 48 h followed by histidine affinity pull-down (pulldown HIS-Ub) and western blotting ( n = 1). INPUT: total cell extracts. TBP was used as control loading in INPUT. Graph shows the quantification of NICD signal in each pulldown fraction relative to the UB-HIS signal in the same fraction. Then, NICD signal in each lane was normalized to the signal in lane 2 (empty vector) set at 100%. For p53, the signal in MDM-395A-expressing cells was normalized to that in lane 3 (MDM2 WT) set at 100%. Lane 1 is the pulldown negative control (without ubiquitin). ( C ) 293 T cells were transfected with NOTCH1-DeltaEx and then co-transfected with 6xtag-His-ubiquitin (UB-HIS) wild-type (WT) or K48R mutant, in combination with empty vector (pcDNA) or MDM2 WT for 48 h, followed by histidine affinity pull-down (pulldown HIS-Ub) and western blotting ( n = 2). INPUT: total cell extracts. TBP was used as loading control in INPUT. Graph shows the quantification of the NICD signal in the pull-down fraction relative to the UB-HIS signal in the same fraction. Then, NICD signal in each lane was normalized to that in lane 3 (MDM2 WT) set at 100%. Lane 1 is the pull-down negative control (without ubiquitin expression). In the legend, n indicates the number of biological replicates. .

Article Snippet: Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and analyzed using the following antibodies against: NICD (#4147, 1:500), phosphorylated ATM (#13050, 1:1000), ATM (#2873S, 1:1000), phosphorylated ATR (#2853, 1:1000), ATR (#2790S, 1:1000), JAG1 (70109S, 1:1000), JAG2 (2210T, 1:1000), DLL3 (78110S 1:1000), DLL4 (96406S, 1:1000), full length NOTCH1 (3608S, 1:1000), GAPDH (2118S, 1:5000), and H3 (59715S, 1:5000) (all from Cell Signaling Technology, Inc) and also DLL1 (QC16739-41705, Antiva Biosciences, 1:1000), MDM2 (#4B2C1.11 and #4B11 Merck Millipore, 1:2000), TUBULIN (#T9026, Sigma, 1:10,000), polyhistidine (H1029 Sigma, 1:1000), p53 (sc-126 Santa Cruz Technology, 1:1000), TBP (TFIID sc-56794, Santa Cruz Technology, 1:500), HSC70 (sc-7298 Santa Cruz Technology, 1:1000), and HEY1 (#19929 Proteintech 1:1000).

Techniques: Transfection, Plasmid Preparation, Western Blot, Cell Fractionation, Control, Ubiquitin Proteomics, Expressing, Negative Control, Mutagenesis

Journal: EMBO Molecular Medicine

Article Title: NOTCH1 intracellular domain stabilization by MDM2 plays a major role in NSCLC response to platinum

doi: 10.1038/s44321-025-00354-9

Figure Lengend Snippet: 293T cells were transfected with NOTCH1-DeltaEx, empty vector (pcDNA), MDM2 WT, MDM2 464 A or MDM2 395 A for 48 h. Chromatin extracts were used for NOTCH1 immunoprecipitation and the levels of the indicated proteins were measured by western blotting. INPUT: total cell extracts. IP ctrl: immunoprecipitation control with an unspecific mouse antibody, IP Notch: immunoprecipitation with an anti-NOTCH1 antibody. TBP was used as loading control in INPUT. .

Article Snippet: Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and analyzed using the following antibodies against: NICD (#4147, 1:500), phosphorylated ATM (#13050, 1:1000), ATM (#2873S, 1:1000), phosphorylated ATR (#2853, 1:1000), ATR (#2790S, 1:1000), JAG1 (70109S, 1:1000), JAG2 (2210T, 1:1000), DLL3 (78110S 1:1000), DLL4 (96406S, 1:1000), full length NOTCH1 (3608S, 1:1000), GAPDH (2118S, 1:5000), and H3 (59715S, 1:5000) (all from Cell Signaling Technology, Inc) and also DLL1 (QC16739-41705, Antiva Biosciences, 1:1000), MDM2 (#4B2C1.11 and #4B11 Merck Millipore, 1:2000), TUBULIN (#T9026, Sigma, 1:10,000), polyhistidine (H1029 Sigma, 1:1000), p53 (sc-126 Santa Cruz Technology, 1:1000), TBP (TFIID sc-56794, Santa Cruz Technology, 1:500), HSC70 (sc-7298 Santa Cruz Technology, 1:1000), and HEY1 (#19929 Proteintech 1:1000).

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Control

Journal: EMBO Molecular Medicine

Article Title: NOTCH1 intracellular domain stabilization by MDM2 plays a major role in NSCLC response to platinum

doi: 10.1038/s44321-025-00354-9

Figure Lengend Snippet: ( A ) Parental and NOTCH1-DeltaEx H358 cells were incubated with doxycycline (0.01 μg/ml) to induce NICD expression (western blot in left panel) and different concentrations of Carboplatin (0, 10, 25 and 50 µM) for 3 days. Then, cell viability was measured ( n = 3). Graph shows the mean ± SEM of three different experiments. P values were obtained by multiple unpaired t test. HSC70 was used as loading control. ( B ) Growth of PDX TP57 tumors in nude mice treated with vehicle ( n = 9), Carboplatin ( n = 8), SP141 (MDM2 inhibitor) + DBZ (Notch inhibitor) ( n = 6), Carboplatin + SP141 ( n = 8), or Carboplatin + DBZ ( n = 8). The y axis shows the tumor volume measured with a caliper. Data are the mean ± SEM. Green P values indicate significant differences between the Carboplatin group and the Carboplatin + DBZ group (two-way ANOVA followed by Tukey’s multiple comparison test). ( C ) Survival analysis in the same animals described in ( B ). The y axis shows the percentage of surviving animals and the x axis the days after treatment. Survival curves were compared with the log rank (Mantel–Cox) test and significant differences were represented by black bold P values for the treated groups compared with the vehicle group and green P value for treated groups compared with the Carboplatin group. ( D ) IHC analysis of γH2AX and CC3 expression levels in PDX TP57 xenografts from nude mice treated with vehicle, Carboplatin alone, Carboplatin + SP141, Carboplatin + DBZ, or SP141 + DBZ ( n = 12 measures from three different tumors for all groups). Data are the mean ± SEM and they correspond to the analysis of 4 fields (original magnification, ×20) per tumor. P values were obtained by one-way ANOVA followed by Tukey’s multiple comparison test. ( E ) The indicated proteins were assessed by western blotting in the same mouse groups as in ( D ) ( n = 2 different tumors/group). In the legend, n indicates the number of biological replicates. .

Article Snippet: Proteins were separated by SDS-PAGE, transferred to PVDF membranes, and analyzed using the following antibodies against: NICD (#4147, 1:500), phosphorylated ATM (#13050, 1:1000), ATM (#2873S, 1:1000), phosphorylated ATR (#2853, 1:1000), ATR (#2790S, 1:1000), JAG1 (70109S, 1:1000), JAG2 (2210T, 1:1000), DLL3 (78110S 1:1000), DLL4 (96406S, 1:1000), full length NOTCH1 (3608S, 1:1000), GAPDH (2118S, 1:5000), and H3 (59715S, 1:5000) (all from Cell Signaling Technology, Inc) and also DLL1 (QC16739-41705, Antiva Biosciences, 1:1000), MDM2 (#4B2C1.11 and #4B11 Merck Millipore, 1:2000), TUBULIN (#T9026, Sigma, 1:10,000), polyhistidine (H1029 Sigma, 1:1000), p53 (sc-126 Santa Cruz Technology, 1:1000), TBP (TFIID sc-56794, Santa Cruz Technology, 1:500), HSC70 (sc-7298 Santa Cruz Technology, 1:1000), and HEY1 (#19929 Proteintech 1:1000).

Techniques: Incubation, Expressing, Western Blot, Control, Comparison

Journal: Journal of Inflammation Research

Article Title: Aryl Hydrocarbon Receptor is Essential in the Control of Lung Club Cell Homeostasis

doi: 10.2147/JIR.S284800

Figure Lengend Snippet: Expression levels of Notch1 signaling molecules are reduced in club cell-specific AhR-null mice. Lung sections were stained for Notch1 (red; (A)) or Hes5 (red; (B)) and CC10 (green) and DAPI (blue)), and the quantification data were showed in ( C ) and (D) (mean ± SEM scores were obtained from 3 animals), respectively. **P < 0.01, ***P < 0.001 ( t tests).

Article Snippet: Western blotting and immunohistochemical (fluorescence) staining analyses were performed as described previously , with the following primary antibodies: actin monoclonal (1:5000 dilution; Sigma–Aldrich), FITC-conjugated anti-goat IgG, rhodamine-conjugated anti-rabbit IgG, alkaline phosphatase-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories), AhR and CC10 goat polyclonal antibodies (Santa Cruz Biotechnology), Notch1 and Hes5 rabbit polyclonal antibodies (cell signaling technology), AhR monoclonal antibody (Enzo life Sciences), and GFP rabbit polyclonal antibody (Abcam).

Techniques: Expressing, Staining

Journal: Journal of Inflammation Research

Article Title: Aryl Hydrocarbon Receptor is Essential in the Control of Lung Club Cell Homeostasis

doi: 10.2147/JIR.S284800

Figure Lengend Snippet: AhR is required for Notch1 mRNA and protein expression in Lung epithelial NL-20 cells; ( A ) NL-20 cells were treated with the AhR ligand Indeno[1,2,3-cd]pyrene (IP) at 1μM, and were examined in Western blotting analyses. Notch1 (NTM) is transmembrane/intracellular protein. ( B ) Doxycycline mediated ectopic AhR expression induces Notch1 in NL-20 cells. ( C ) IP-treated AhR-null cells showed lower expression levels of NOTCH1 and the downstream gene HES5 in NL-20 cells. ( D ) IP-treated (1μM) AhR-null cells had attenuated Notch1 (NTM) expression in NL-20 cells. ( E ) Serial deletion constructs of Notch1 promoters for promoter-reporter assays; ( F ) A549 cells were transfected with a series of deletion constructs of the Notch1 promoter and luciferase activities were determined. ( G ) A549 cells with ectopic AhR-GFP expression were analyzed in chromatin immunoprecipitation assays with IgG and GFP antibodies; *P < 0.05, **P < 0.01, ****P < 0.0001 ( t tests or One-Way ANOVA).

Article Snippet: Western blotting and immunohistochemical (fluorescence) staining analyses were performed as described previously , with the following primary antibodies: actin monoclonal (1:5000 dilution; Sigma–Aldrich), FITC-conjugated anti-goat IgG, rhodamine-conjugated anti-rabbit IgG, alkaline phosphatase-conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories), AhR and CC10 goat polyclonal antibodies (Santa Cruz Biotechnology), Notch1 and Hes5 rabbit polyclonal antibodies (cell signaling technology), AhR monoclonal antibody (Enzo life Sciences), and GFP rabbit polyclonal antibody (Abcam).

Techniques: Expressing, Western Blot, Construct, Transfection, Luciferase, Chromatin Immunoprecipitation

Journal: Journal of the American Heart Association

Article Title: Phospholipase Cγ1 Mediates Intima Formation Through Akt‐Notch1 Signaling Independent of the Phospholipase Activity

doi: 10.1161/jaha.117.005537

Figure Lengend Snippet: Figure 1. Phospholipase Cc1 (PLCc1) is required for Notch1 cleavage stimulated by angiotensin II (Ang II) and platelet-derived growth factor (PDGF). A through D, Rat aortic smooth muscle cells (RASMCs) were treated with 200 nmol/L Ang II for different time (A and C) or different dose of Ang II for 1 hour (B and D). Notch1 intracellular domain (N1-ICD), FL-Notch1, and b-actin expression were measured by Western blot. Notch1 cleavage (measured by N1-ICD/Notch1) after Ang II stimulation was quantified (meanSEM, n=3) (C and D). *P<0.05 vs the cells not treated with Ang II. E through H, RASMCs were treated with 20 ng/mL of PDGF for different time (E and G) or different dose of PDGF for 1 hour (F and H). N1-ICD, FL-Notch1, and b-actin expression were measured by Western blot. Notch1 cleavage (measured by N1- ICD/Notch1) after PDGF stimulation was quantified (meanSEM) (G and H). *P<0.05 vs the cells not treated with PDGF. I and J, RASMCs were transfected with control small interfering RNA (siCtr) or PLCc1 small interfering RNA (siPLCc1) for 48 hours followed by stimulation of Ang II (200 nmol/L, E) or PDGF (20 ng/mL, F) for 1 hour. N1-ICD, FL-Notch1, N2-ICD, FL-Notch2, t-PLCc1, and b-actin expression were measured by Western blot. K, PLCc1 protein levels were quantified (normalized to b-actin after small interfering RNA knockdown) (meanSEM, n=3). #P<0.05 vs the siCtr group. L and M, Notch1 cleavage was quantified by N1-ICD normalized to FL-Notch1 (meanSEM, n=3). *P<0.05 vs the siCtr group. #P<0.05 vs the siCtr+PDGF group. Experiments were performed in triplicate.

Article Snippet: Primary antibodies of phospho-Akt Ser473, phospho-Akt Thr308, Akt, phosphoPLCc1 Tyr783, PLCc1, 3-phosphoinositide-dependent protein kinase-1 (PDK1), mammalian target of rapamycin (mTOR), and Notch2 were purchased from Cell Signaling Technology; Hey2 antibody was purchased from Beijing Biosynthesis Biotechnology; antibody of Notch1 used in Western blot was from Santa Cruz Biotechnology; and Notch1 intracellular domain (N1-ICD) antibody used in immunohistochemistry was from Millipore.

Techniques: Derivative Assay, Expressing, Western Blot, Transfection, Control, Small Interfering RNA, Knockdown

Journal: Journal of the American Heart Association

Article Title: Phospholipase Cγ1 Mediates Intima Formation Through Akt‐Notch1 Signaling Independent of the Phospholipase Activity

doi: 10.1161/jaha.117.005537

Figure Lengend Snippet: Figure 3. Phospholipase Cc1 (PLCc1)–mediated Akt activation is essential for Notch1 cleavage. A through G, Representative immunoblot and quantification of Akt phosphorylation (p-Akt) on S473 and T308 after PLCc1 depletion. Rat aortic smooth muscle cells (RASMCs) were transfected with control small interfering RNA (siCtr) or PLCc1 small interfering RNA (siPLCc1) for 48 hours and then stimulated by angiotensin II (Ang II; 200 nmol/L, A, D, and E) or platelet-derived growth factor (PDGF; 20 ng/mL, B, F, and G) for 5 minutes. PLCc1 levels were quantified after small interfering RNA (siRNA) knockdown (normalized to b-actin) (C). P-Akt phosphorylation on S473 and T308 were quantified after small interfering RNA knockdown (normalized to total Akt) (D through G). Bars indicate meanSEM. *P<0.05 vs the siCtr group. #P<0.05 vs the siCtr with Ang II or PDGF treatment. H, The binding of PLCc1 and Akt was assayed by immunoprecipitation (IP) with t- Akt antibody and probing for t-PLCc1. Serum-starved RASMCs were treated with vehicle, 200 nmol/L Ang II, or 20 ng/mL PDGF for 5 minutes. Then cell lysate was collected for IP assay. I and J, The binding of PLCc1 and mammalian target of rapamycin (mTOR) (I) or 3- phosphoinositide-dependent protein kinase-1 (PDK1) (J) was assayed by IP with mTOR antibody or PDK1 antibody and probing for PLCc1 in RASMCs. K through M, Akt activation after overexpression of phospholipase site–mutated PLCc1 (H335F and H380F) was assayed in PLCc1 knockdown human embryonic kidney 293T cells. Effects of Akt or Notch inhibitors on PLCc1 and p-Akt and Notch1 cleavage induced by Ang II (N) or PDGF (O). RASMCs were pretreated with 1 lmol/L wortmannin (Wort) and 10 lmol/L N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S- phenylglycine t-butyl ester (DAPT) for 12 hours and then incubated with Ang II (200 nmol/L) or PDGF (20 ng/mL). All experiments were performed in triplicate. DMSO indicates dimethyl sulfoxide; WT, wild-type; MUT, mutant.

Article Snippet: Primary antibodies of phospho-Akt Ser473, phospho-Akt Thr308, Akt, phosphoPLCc1 Tyr783, PLCc1, 3-phosphoinositide-dependent protein kinase-1 (PDK1), mammalian target of rapamycin (mTOR), and Notch2 were purchased from Cell Signaling Technology; Hey2 antibody was purchased from Beijing Biosynthesis Biotechnology; antibody of Notch1 used in Western blot was from Santa Cruz Biotechnology; and Notch1 intracellular domain (N1-ICD) antibody used in immunohistochemistry was from Millipore.

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Transfection, Control, Small Interfering RNA, Derivative Assay, Knockdown, Binding Assay, Immunoprecipitation, Over Expression, Incubation, Mutagenesis

Journal: Journal of the American Heart Association

Article Title: Phospholipase Cγ1 Mediates Intima Formation Through Akt‐Notch1 Signaling Independent of the Phospholipase Activity

doi: 10.1161/jaha.117.005537

Figure Lengend Snippet: Figure 6. Phospholipase Cc1 (PLCc1) is critical for Akt and Notch1 activation in the carotid artery after ligation. A, Representative immunoblot of Akt phosphorylation (p-Akt) in carotid arteries transfected with control small interfering RNA (siCtr) or PLCc1 small interfering RNA (siPLCc1) 7 days after ligation. B, Quantification of PLCc1 protein levels after small interfering RNA knockdown on day 7 (normalized to b-actin) (meanSEM). C, Quantification of p-Akt in carotid arteries on day 7 (meanSEM, n=5). D, Immunostaining of Notch1 intracellular domain (N1-ICD) in ligated carotid arteries on day 21. Red arrows indicate N1-ICD–positive cells. E, N1-ICD–positive cells were quantified by examining every section of 5 sections in each artery (meanSEM, n=5). F, Immunohistochemistry of Hey2 in carotid arteries on day 21. G, Hey2 expression was quantified by examining every section of 5 sections in each artery (meanSEM, n=5). *P<0.05 compared with the siCtr group. #P<0.05 compared with the siCtr+PDGF group in (B, D, and F).

Article Snippet: Primary antibodies of phospho-Akt Ser473, phospho-Akt Thr308, Akt, phosphoPLCc1 Tyr783, PLCc1, 3-phosphoinositide-dependent protein kinase-1 (PDK1), mammalian target of rapamycin (mTOR), and Notch2 were purchased from Cell Signaling Technology; Hey2 antibody was purchased from Beijing Biosynthesis Biotechnology; antibody of Notch1 used in Western blot was from Santa Cruz Biotechnology; and Notch1 intracellular domain (N1-ICD) antibody used in immunohistochemistry was from Millipore.

Techniques: Activation Assay, Ligation, Western Blot, Phospho-proteomics, Transfection, Control, Small Interfering RNA, Knockdown, Immunostaining, Immunohistochemistry, Expressing

Journal: Journal of the American Heart Association

Article Title: Phospholipase Cγ1 Mediates Intima Formation Through Akt‐Notch1 Signaling Independent of the Phospholipase Activity

doi: 10.1161/jaha.117.005537

Figure Lengend Snippet: Figure 7. Proposed model of phospholipase Cc1 (PLCc1)–mediated Akt-Notch1 signaling in vascular smooth muscle cell (VSMC) function. After vessel injury, angiotensin II (Ang II), and platelet-derived growth factor (PDGF) are excessively released and activate their receptors on VSMC, and then induce PLCc1 phosphorylation. Phosphorylated PLCc1 recruits 3-phosphoinositide-dependent protein kinase-1 (PDK1) and mammalian target of rapamycin complex 2 (mTORC2) to form PLCc1-PDK1-Akt or PLCc1-mTOR-Akt complexes and promotes Akt phosphorylation. Activated Akt facilitates Notch1 cleavage and Notch1 intracellular domain (N1-ICD) translocates to the nucleus, inducing the expression of Hey2 and other target genes. These transduction cascades promote VSMC proliferation, migration, survival, and dedifferentiation, leading to intima formation. AT1R indicates Ang II receptor 1; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor.

Article Snippet: Primary antibodies of phospho-Akt Ser473, phospho-Akt Thr308, Akt, phosphoPLCc1 Tyr783, PLCc1, 3-phosphoinositide-dependent protein kinase-1 (PDK1), mammalian target of rapamycin (mTOR), and Notch2 were purchased from Cell Signaling Technology; Hey2 antibody was purchased from Beijing Biosynthesis Biotechnology; antibody of Notch1 used in Western blot was from Santa Cruz Biotechnology; and Notch1 intracellular domain (N1-ICD) antibody used in immunohistochemistry was from Millipore.

Techniques: Derivative Assay, Phospho-proteomics, Expressing, Transduction, Migration

Journal: Cell

Article Title: Dynamic Ligand Discrimination in the Notch Signaling Pathway

doi: 10.1016/j.cell.2018.01.002

Figure Lengend Snippet: (A) C2C12 cells were engineered to expressed Notch1 receptors lacking the extracellular domain (N1DECD, green). This receptor is inactive in the presence of the γ-secretase inhibitor DAPT (red), but constitutively active when DAPT concentration is reduced in the culture medium. (B) Comparison of transcript levels in C2C12-N1ΔECD cells at 1 hr or 6 hr after DAPT removal. The blue line represents equal expression at 1 hr and 6 hr, and the gray lines represent 5-fold changes in either direction. Circled genes are putative direct Notch targets. The blue circle highlights target genes that are upregulated >5-fold at 1 hr but not 6 hr, while red circles indicate target genes that are upregulated >5-fold only after 6 hr. See also and . (C) qPCR time course measurement of Hes1 (blue), Hey1 (orange), and HeyL (yellow) mRNA levels following complete DAPT removal at t = 0 hr. (D) Duration dependence of Hes1 (blue) and Hey1 (orange) response to DAPT removal for 5 min, 15 min, or 30 min followed by replenishment (“Pulse”), or no replenishment until the 1 hr or 4 hr measurement (“Sustained”). Error bars represent SEM calculated from duplicate experiments (n = 2). See also Figure S4 .

Article Snippet: Cells were then incubated with recombinant mouse Notch1 ext -Fc protein in binding solution (blocking solution containing 100 ug/ml CaCl 2 , R&D Systems) for 45 min at RT.

Techniques: Concentration Assay, Comparison, Expressing

Journal: Cell

Article Title: Dynamic Ligand Discrimination in the Notch Signaling Pathway

doi: 10.1016/j.cell.2018.01.002

Figure Lengend Snippet: (A) Both Dll1 (blue) and Dll4 (red) activate the Notch1 receptor (green) to induce proteolytic release of the Notch intracellular domain (NICD), but are used in different biological contexts (blue and red boxes, bottom). The released NICD translocates to the nucleus and, in complex with CSL/RBPjκ (yellow), activates Notch target genes (white). (B) Left: Engineered CHO-K1 “sender” cell lines contain stably integrated constructs expressing Dll1 (blue) or Dll4 (red), each with a co-translational (T2A, brown) H2B-mCh readout (purple), from a 4epi-Tetracycline (4epi-Tc) inducible promoter. Right: “Receiver” cells stably express a chimeric receptor combining the Notch1 extracellular domain (Notch1ECD) with a Gal4 transcription factor (orange), which can activate a stably integrated fluorescent H2B-3xCitrine reporter gene (chartreuse). (C) Left (schematics): A minority of receiver cells (green) are co-cultured with an excess of either Dll1 (blue) or Dll4 (red) sender cells. Right: Filmstrips showing representative sustained (top, Dll4 senders) or pulsatile (bottom, Dll1 senders) response of a single receiver cell (center, automatically segmented nucleus outlined in white). Grey channel shows DIC images of cells, while the rate of increase in Citrine fluorescence, scaled to 25%–75% of its total range, is indicated using green pseudo-coloring. See also and . (D) Left: Representative traces showing total nuclear Citrine fluorescence levels (top) or corresponding derivatives of the total Citrine ( d Citrine/ d t), i.e., promoter activity (bottom), in individual receiver cells activated by Dll4. Right: Average values of total fluorescence (top) and promoter activity (bottom) in receiver cells activated by Dll4. Solid traces represent medians, lighter shades indicate SEM, and gray shading indicates SD. n, number of traces included in the alignment. See for alignment and normalization procedure. (E) Left: Corresponding plots (as in D) showing total nuclear Citrine fluorescence levels (top) and promoter activity (bottom) in individual receiver cells in co-culture with Dll1. Right: Average values of total fluorescence (top) and promoter activity (bottom) in receiver cells activated by Dll1. The percentage value (60%) in the plots on right indicates the fraction of receiver traces included in the alignment ( STAR Methods , see also ). (F) 95 th percentile of (absolute, non-normalized) promoter activity values between 0 and 7.5 hr (after alignment) in the traces included in (D) and (E). This time window is chosen to simultaneously estimate the promoter activity at the peak of Dll1 pulses and at steady-state levels of Dll4 signaling. Solid horizontal lines represent medians, while the boxes delineate 25 th –75 th percentile values. p value calculated by two-sided Kolmogorov-Smirnov (K-S) test. See also Figures S1 and S2 .

Article Snippet: Cells were then incubated with recombinant mouse Notch1 ext -Fc protein in binding solution (blocking solution containing 100 ug/ml CaCl 2 , R&D Systems) for 45 min at RT.

Techniques: Stable Transfection, Construct, Expressing, Cell Culture, Fluorescence, Activity Assay, Co-Culture Assay

Journal: Cell

Article Title: Dynamic Ligand Discrimination in the Notch Signaling Pathway

doi: 10.1016/j.cell.2018.01.002

Figure Lengend Snippet: (A) Developing chick embryo (dorsal view schematic). Dll1 (blue cells in 3) is expressed in a fraction of neural crest cells (gray, see 2, 3). These cells activate Notch1-expressing Pax7 + progenitor cells in the dorsomedial lip (DML, magenta) of the somite. When activated, these progenitor cells (green, 3) upregulate Hes1 and the muscle regulatory gene MyoD1. (B–D) Representative images showing effects of Dll1 or Dll4 electroporation into the neural crest, on Hes1, Hey1, and MyoD1 expression in the DML. White arrows indicate the somites on the electroporated side. The dotted lines indicate the DMLs of somites or the central line of the neural tube. (B) Top: Dll1-T2A-EGFP (i, blue), electroporated into the left side of the neural tube, is expressed in the neural tube and neural crest, resulting in upregulation of Hes1 (ii, red) and MyoD1 (iii, green) in the somites on the electroporated (left) side compared to the right side, which serves as negative control. Bottom: When Dll4-T2A-EGFP (iv, blue) is electroporated, Hey1 (v, red) is upregulated on the electroporated side, and MyoD1 (vi, green) expression is decreased. (C) Dll1-T2A-EGFP (blue, left) electroporation does not affect expression of Hey1 (red, right) in adjacent somites. (D) Dll4-T2A-EGFP (blue, left) electroporation increases expression of Hes1 (red, right) in adjacent somites. See also and .

Article Snippet: Cells were then incubated with recombinant mouse Notch1 ext -Fc protein in binding solution (blocking solution containing 100 ug/ml CaCl 2 , R&D Systems) for 45 min at RT.

Techniques: Expressing, Electroporation, Negative Control

Journal: Cell

Article Title: Dynamic Ligand Discrimination in the Notch Signaling Pathway

doi: 10.1016/j.cell.2018.01.002

Figure Lengend Snippet: (A and B) Dll4 ECD -Dll1 ICD and Dll1 ECD -Dll4 ICD were constructed by exchanging the intracellular domain (ICD) of Dll4 with that of Dll1. (A) Median response profiles in activated receiver cells co-cultured with Dll4 sender cells (red, top left) or Dll4 ECD -Dll1 ICD sender cells (magenta, right) under excess receiver conditions (as in ). Solid traces represent medians, lighter colored regions represent SEM, and gray shading represents SD. n, number of cell traces included in the alignment. See for alignment and normalization procedures. Bottom left: 95 th percentile of (absolute, non-normalized) promoter activity values between 0 and 7.5 hr (after alignment) in individual traces included in the averaging. Solid horizontal lines represent medians, while the boxes delineate 25 th –75 th percentile values. p value calculated by two-sided K-S test. (B) Corresponding response profiles (right, top left) and amplitudes (bottom left) in activated receiver cells co-cultured with Dll1 sender cells (blue) or Dll1 ECD- Dll4 ICD sender cells (purple) under excess sender conditions. (C) Representative images of “excess sender” co-cultures of receiver cells (R) expressing full-length Notch1 and sender cells (S) expressing either Dll4 ECD -Dll1 ICD (left) or Dll4 (Dll4 ECD -Dll4 ICD , right), immunostained for Notch1ECD. Examples of dispersed, low intensity staining or higher-intensity puncta are indicated by the white circles. (D) Left: Median values of number of puncta detected (see ) in Dll1 ICD (blue) or Dll4 ICD (red) sender cells neighboring receiver cells. Right: Median values of the (background subtracted) mean pixel intensity of dispersed signal (see ) within Dll1 ICD (blue) or Dll4 ICD (red) sender cells that neighbor receiver cells. Error bars represent SEM. p value calculated using the two-sided K-S test. (E) Schematic: Proposed differences in the abilities of ligands containing the Dll1 (blue) and Dll4 (red) ICDs to initiate transendocytosis in different clustering states. See also Figure S6 .

Article Snippet: Cells were then incubated with recombinant mouse Notch1 ext -Fc protein in binding solution (blocking solution containing 100 ug/ml CaCl 2 , R&D Systems) for 45 min at RT.

Techniques: Construct, Cell Culture, Activity Assay, Expressing, Staining