Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Development and epigenetic plasticity of murine Müller glia

doi: 10.1016/j.bbamcr.2019.06.019

Figure Lengend Snippet: List of oligonucleotides and antibodies used in this study

Article Snippet: Isolation of Notch1+ cells, Glast+ cells, and cortical astrocytes We used the same protocol for immunomagnetic cell separation described in Dvoriantchikova et al. to isolate Notch1+ cells with monoclonal biotin-conjugated anti-Notch1 mouse antibodies (130–096-557, Miltenyi Biotec, Auburn, CA) [ 18 ].

Techniques:

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Development and epigenetic plasticity of murine Müller glia

doi: 10.1016/j.bbamcr.2019.06.019

Figure Lengend Snippet: Quantitative RT-PCR analysis has confirmed the microarray data for a group of selected genes. For each gene, the results are expressed as percentages ± SEM of the corresponding values in the Notch1+ cells isolated from P0 developing retinas (P0 Notch1+ cells).

Article Snippet: Isolation of Notch1+ cells, Glast+ cells, and cortical astrocytes We used the same protocol for immunomagnetic cell separation described in Dvoriantchikova et al. to isolate Notch1+ cells with monoclonal biotin-conjugated anti-Notch1 mouse antibodies (130–096-557, Miltenyi Biotec, Auburn, CA) [ 18 ].

Techniques: Quantitative RT-PCR, Microarray, Isolation

Journal: Biochimica et biophysica acta. Molecular cell research

Article Title: Development and epigenetic plasticity of murine Müller glia

doi: 10.1016/j.bbamcr.2019.06.019

Figure Lengend Snippet: The results of the hierarchical and k-means clustering revealed the changes that are undergone in RPCs (Notch1+ cells) and Müller glia (Glast+ cells) during retinal development. A) Hierarchical clustering of differentially expressed genes showed that many P7 and P14 Notch1+ cells (late RPCs) are predisposed to become Müller glia. While Glast+ cells at P7 are still very close to a multipotent progenitor state, P14, P21, and P28 Glast+ cells are already grouped together indicating the adult Müller glia state. B) The 10 clusters were identified by the k-means clustering algorithm. C) The majority of differentially expressed genes were located in cluster 1, 3, and 4. D) Meanwhile, significant changes were undergone in genes located in small clusters 5, 7, and 8. A value F (F statistic) was calculated using the ANOVA test.

Article Snippet: Isolation of Notch1+ cells, Glast+ cells, and cortical astrocytes We used the same protocol for immunomagnetic cell separation described in Dvoriantchikova et al. to isolate Notch1+ cells with monoclonal biotin-conjugated anti-Notch1 mouse antibodies (130–096-557, Miltenyi Biotec, Auburn, CA) [ 18 ].

Techniques:

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: 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