Journal: Aging (Albany NY)

Article Title: Long non-coding RNA RP11-59H7.3 promotes cell proliferation and invasion metastasis in colorectal cancer by miR-139-5p/NOTCH1 axis

doi: 10.18632/aging.103331

Figure Lengend Snippet: miR-139-5p is directly targeted by RP11-59H7.3. ( A ) miR-139-5p-binding sequence in NOTCH1 3'UTR and RP11-59H7.3. The mutation was induced in RP11-59H7.3 in the site complementary to the miR-139-5p binding. ( B ) RT-qPCR was performed to determine levels of miRNA in cells of SW480 post-transfection by pcDNA-RP11-59H7.3. The activity of luciferase in cells of 293T co-transfected by miR-139-5p ( C ) or miR-203 ( D ) mimics and the luciferase reporters (mutant RP11-59H7.3) or control (wild type RP11-59H7.3). The activity of renilla luciferase was determined then normalized to firefly luciferase activity level. Each experiment was repeated thrice, and the data are summarized as mean ± SD (two-tailed Student’s t-test). ** p < 0.01; * p < 0.05.

Article Snippet: We procured the following antibodies NOTCH1 (1:1000, 4380T, CST), HES1(1:1000, 11988S, CST), HEY1(1:1000, ab22614, Abcam), Bax (1:2000, ab182733, Abcam), Bcl-2(1:1000, ab32124, Abcam), E-cadherin(1:1000, 5296S, CST), Vimentin(1:1000, 5741T, CST), GAPDH (1:1000, ab9484, Abcam), from Cell Signaling Technology and Abcam (China) for use in the study.

Techniques: Binding Assay, Sequencing, Mutagenesis, Quantitative RT-PCR, Transfection, Activity Assay, Luciferase, Control, Two Tailed Test

Journal: Aging (Albany NY)

Article Title: Long non-coding RNA RP11-59H7.3 promotes cell proliferation and invasion metastasis in colorectal cancer by miR-139-5p/NOTCH1 axis

doi: 10.18632/aging.103331

Figure Lengend Snippet: RP11-59H7.3 sponges miR-139-5p and moderate expression of NOTCH1. ( A ) The activity of luciferase of a luciferase reporter vector (pLuc) harboring mutant RP11-59H7.3 or wild-type co-transfected by miR-139-5p was evaluated using the dual luciferase assay. ( B ) cell lysates of SW480 cells were used for RIP with an IgG antibody or anti-Ago2 antibody. The miR-139-5p and RP11-59H7.3 levels were analyzed using RT-qPCR. ( C ) pLuc plasmid and miR-139-5p harboring NOTCH1 3'UTRs were co-transfected by pcDNA-RP11-59H7.3 or empty vector into cells of 293T to determine whether RP11-59H7.3 could act as a ceRNA of miR-139-5p. ( D , G ) The NOTCH1 expression levels in cells of SW480 prior transfected by pcDNA-RP11-59H7.3 and LoVo cells prior transfected by sh-lncRNA-RP11-59H7.3 were determined using western blot and RT-qPCR. ( E , F ) Analysis of correlation between expression of RP11-59H7.3/miR-139-5p and RP11-59H7.3/NOTCH1. ** p < 0.01; * p < 0.05.

Article Snippet: We procured the following antibodies NOTCH1 (1:1000, 4380T, CST), HES1(1:1000, 11988S, CST), HEY1(1:1000, ab22614, Abcam), Bax (1:2000, ab182733, Abcam), Bcl-2(1:1000, ab32124, Abcam), E-cadherin(1:1000, 5296S, CST), Vimentin(1:1000, 5741T, CST), GAPDH (1:1000, ab9484, Abcam), from Cell Signaling Technology and Abcam (China) for use in the study.

Techniques: Expressing, Activity Assay, Luciferase, Plasmid Preparation, Mutagenesis, Transfection, Quantitative RT-PCR, Western Blot

Journal: Aging (Albany NY)

Article Title: Long non-coding RNA RP11-59H7.3 promotes cell proliferation and invasion metastasis in colorectal cancer by miR-139-5p/NOTCH1 axis

doi: 10.18632/aging.103331

Figure Lengend Snippet: RP11-59H7.3 promotes CRC cell migration and invasion through miR-139-5p/NOTCH1 axis. ( A , B ) wound-healing assays were performed in LoVo and SW480 cells prior transfected by miR-139-5p mimic or co-transfected by pcDNA-RP11-59H7.3 and miR-139-5p mimic, and calculations of relative gap distance were completed and a histogram plotted. ( C , D ) cell movement and invasion assays were applied to determine the invasion ability and vertical migration of colorectal cancer cells, and the cell number was calculated and represented on a histogram. ( E ) Western blot was performed to determine the metastasis-related protein expression in colorectal cancer cells. ( F ) Relative NOTCH1 protein levels in colorectal cells after RP11-59H7.3 overexpression and Mimics of miR-139-5p. ** p < 0.01; * p < 0.05.

Article Snippet: We procured the following antibodies NOTCH1 (1:1000, 4380T, CST), HES1(1:1000, 11988S, CST), HEY1(1:1000, ab22614, Abcam), Bax (1:2000, ab182733, Abcam), Bcl-2(1:1000, ab32124, Abcam), E-cadherin(1:1000, 5296S, CST), Vimentin(1:1000, 5741T, CST), GAPDH (1:1000, ab9484, Abcam), from Cell Signaling Technology and Abcam (China) for use in the study.

Techniques: Migration, Transfection, Western Blot, Expressing, Over Expression

Journal: Aging (Albany NY)

Article Title: Long non-coding RNA RP11-59H7.3 promotes cell proliferation and invasion metastasis in colorectal cancer by miR-139-5p/NOTCH1 axis

doi: 10.18632/aging.103331

Figure Lengend Snippet: The sequences of shRNA for RP11-59H7.3.

Article Snippet: We procured the following antibodies NOTCH1 (1:1000, 4380T, CST), HES1(1:1000, 11988S, CST), HEY1(1:1000, ab22614, Abcam), Bax (1:2000, ab182733, Abcam), Bcl-2(1:1000, ab32124, Abcam), E-cadherin(1:1000, 5296S, CST), Vimentin(1:1000, 5741T, CST), GAPDH (1:1000, ab9484, Abcam), from Cell Signaling Technology and Abcam (China) for use in the study.

Techniques: shRNA

Journal: Nature Communications

Article Title: SLC35D3 promotes white adipose tissue browning to ameliorate obesity by NOTCH signaling

doi: 10.1038/s41467-023-43418-5

Figure Lengend Snippet: a KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis results of IngWAT from WT and SAKI mice ( n = 3 per group). P values are determined using the clusterProfiler R package’s built-in hypergeometric test, and adjusted P values are calculated using Benjamini–Hochberg correction. b GSEA (gene set enrichment analysis) results showed that the Notch signaling pathway was suppressed in the IngWAT of SAKI mice ( n = 3 per group). The P values in GSEA are calculated empirically with permutation test by permuting the gene labels at random according to the GSEA algorithm, and FDR q values (corrected P values) are calculated by Benjamini–Hochberg FDR correction. The NES and FDR q values are shown in the images. NES, normalized enrichment score; FDR, false positive rate. c , e qPCR analysis of Slc35d3 and Notch1-related genes in IngWAT from SAKI mice and WT mice ( c ), as well as in IngWAT from SAKO mice and Flox mice ( e ); n = 6 per group. d , f Western blot showing the expression of Notch1-related proteins in IngWAT from SAKI mice and their controls ( d ), as well as in IngWAT from SAKO mice and their controls ( f ); n = 3 per group. Right panel, summary of the quantification of three independent experiments. g qPCR detection of NOTCH1 expression levels in HEK293T cells overexpressing SLC35D3 ; n = 6 per group. h Western blot analysis of NOTCH1 protein in HEK293T cells overexpressing SLC35D3 . Right panel, summary of the quantification of three independent experiments. i Western blot analysis of NOTCH1 protein in HEK293T cells treated with CHX (cycloheximide). Right panel, summary of the quantification of three independent experiments. Values are the fold induction of gene expression normalized to the housekeeping gene β-Actin ( c – i ). Data are expressed as the mean ± SEM. Statistical significance was assessed by unpaired two-sided Student’s t test ( c – i ). The exact P values are shown in the figure. SAKI Slc35d3 adipocyte-specific knock-in, IngWAT inguinal white adipose tissue. Source data are provided as a Source Data file.

Article Snippet: Then, the cells were incubated with PE-conjugated Notch1 antibody (CST, 15004) at room temperature for 1 h with rotation, and washed twice.

Techniques: Western Blot, Expressing, Gene Expression, Knock-In

Journal: Nature Communications

Article Title: SLC35D3 promotes white adipose tissue browning to ameliorate obesity by NOTCH signaling

doi: 10.1038/s41467-023-43418-5

Figure Lengend Snippet: a , b CoIP assay results showed the interaction between SLC35D3 and NOTCH1 ( a ), as well as the interaction between SLC35D3 and ECD ( b ). Representative images from three independent experiments are shown. c Direct interaction between SLC35D3 and ECD was further confirmed by pull-down assay. Representative images from three independent experiments are shown. d , e SLC35D3 overexpression promoted the colocalization of NOTCH1 and ER. Representative confocal images of cells are shown ( d ), as well as the quantification results of four independent parallel experiments ( e ). The inset in the right panel shows a magnified view of the indicated area. Scale bar, 20 μm. CALNEXIN, an ER marker protein. f , g SAKO mice showed higher levels of Notch1 at the cell membrane in IngWAT than control mice. Representative confocal images are shown ( f ), as well as the quantification results of six independent parallel experiments ( g ). Scale bar, 50 μm. Na-K ATPase, a plasma membrane marker protein. h – j Membrane proteins and endoplasmic reticulum proteins of IngWAT were isolated from SAKO and SAKI mice ( n = 3 per group). The western blot results are shown ( h ), as well as the quantitative analysis results ( i , j ). Total NOTCH1 expression was normalized to β-Actin expression, membrane NOTCH1 expression was normalized to Na-K ATPase, and ER NOTCH1 expression was normalized to CALNEXIN. k – m Primary adipocytes were isolated from the IngWAT of SAKO mice and SAKI mice ( n = 3 per group). Cell surface biotinylation assay results are shown ( k ), as well as the quantitative analysis results ( l , m ). Data are expressed as the mean ± SEM. Statistical significance was assessed by unpaired two-sided Student’s t test ( e , g , i , j , l , m ). The exact P values are shown in the figure. SAKO Slc35d3 adipocyte-specific knockout, SAKI Slc35d3 adipocyte-specific knock-in, OE overexpression, IngWAT inguinal white adipose tissue, ER endoplasmic reticulum. Source data are provided as a Source Data file.

Article Snippet: Then, the cells were incubated with PE-conjugated Notch1 antibody (CST, 15004) at room temperature for 1 h with rotation, and washed twice.

Techniques: Co-Immunoprecipitation Assay, Pull Down Assay, Over Expression, Marker, Membrane, Control, Clinical Proteomics, Isolation, Western Blot, Expressing, Cell Surface Biotinylation Assay, Knock-Out, Knock-In

Journal: Nature Communications

Article Title: SLC35D3 promotes white adipose tissue browning to ameliorate obesity by NOTCH signaling

doi: 10.1038/s41467-023-43418-5

Figure Lengend Snippet: a , k Illustration of Notch1 knockdown in the IngWAT of SAKO ( a ) and SAKI ( k ) mice. b , l Notch1 was specifically knocked down in the IngWAT of SAKO ( b ) and SAKI ( l ) mice; n = 4 per group. c , m Body weights of NCD-fed SAKO mice ( c ) and HFD-fed SAKI mice ( m ); n = 4 per group. d , e , n , o GTT ( d ) and ITT ( e ) results of NCD-fed SAKO mice ( n = 4 per group), as well as GTT ( n ) and ITT ( o ) results of HFD-fed SAKI mice ( n = 4 per group). The area under the curve (AUC) values are shown in the right panel. f , g , p , q Serum TG and TC levels in the NCD-fed SAKO mice ( f , g ) and HFD-fed SAKI mice ( p , q ); n = 4 per group. h , r Lipolytic gene expression levels of IngWAT in the NCD-fed SAKO mice ( h ) and HFD-fed SAKI mice ( r ); n = 4 per group. i , s Thermogenic gene expression levels of IngWAT in the NCD-fed SAKO mice ( i ) and HFD-fed SAKI mice ( s ); n = 4 per group. j , t HE staining results and adipocyte size quantification of IngWAT in the NCD-fed SAKO mice ( j ) and HFD-fed SAKI mice ( t ). Representative images of HE staining results from three independent experiments are shown. Right panel, the quantitative results of the size of each adipocyte counted ( j : shCtrl, n = 104 cells; shNotch1, n = 107 cells. t : shCtrl, n = 102 cells; shNotch1, n = 101 cells). Scale bar, 20 μm. Values are the fold induction of gene expression normalized to the housekeeping gene β-Actin ( b , h , i , l , r , s ). Data are expressed as the mean ± SEM. Statistical significance was assessed by unpaired two-sided Student’s t test ( b , c , f – k , l , m , p – t ) or two-way ANOVA followed by Bonferroni’s multiple comparisons test ( d , e , n , o ). The exact P values are shown in the figure. SAKO Slc35d3 adipocyte-specific knockout, SAKI Slc35d3 adipocyte-specific knock-in, IngWAT inguinal white adipose tissue, TG triglyceride, TC total cholesterol, NCD normal chow diet, HFD high-fat diet. Source data are provided as a Source Data file.

Article Snippet: Then, the cells were incubated with PE-conjugated Notch1 antibody (CST, 15004) at room temperature for 1 h with rotation, and washed twice.

Techniques: Knockdown, Gene Expression, Staining, Knock-Out, Knock-In

Journal: Nature Communications

Article Title: SLC35D3 promotes white adipose tissue browning to ameliorate obesity by NOTCH signaling

doi: 10.1038/s41467-023-43418-5

Figure Lengend Snippet: SLC35D3 regulates obesity by interacting with the extracellular domain (ECD) of NOTCH1, which promotes NOTCH1 accumulation in the endoplasmic reticulum (ER) and inhibits NOTCH1 transport to the cell membrane, ultimately inhibiting NOTCH1 signaling. IngWAT, inguinal white adipose tissue.

Article Snippet: Then, the cells were incubated with PE-conjugated Notch1 antibody (CST, 15004) at room temperature for 1 h with rotation, and washed twice.

Techniques: Membrane

Journal: Nagoya Journal of Medical Science

Article Title: XXYLT1 inhibits NOTCH1 activation in Jurkat cells while promoting cell proliferation

doi: 10.18999/nagjms.87.3.431

Figure Lengend Snippet: NOTCH1 EGF repeats overexpressed in Jurkat cells are modified with the O- glucose trisaccharide at EGF10 Fig. 1A: HCD-MS/MS spectra of a glycopeptide from NOTCH1EGF10 carrying the O- Glc trisaccharide Xyl-Xyl-Glc. Numerous b and y ions in addition to the sequential neutral loss of sugar residues from the precursor ion confirm the identity of the peptide and the presence of the O- Glc trisaccharide. Fig. 1B–E: EICs of glycopeptides from EGF10 modified with previously reported glycoforms. Blue circles, glucose; Orange stars, Xyl; White circles, uncharacterized hexose; Black dashed line, naked peptide; Blue, peptide + O- Glc; Yellow, peptide + O- Glc-Xyl; Orange, peptide + O- Glc-Xyl-Xyl; Gray, peptide + 2 hexoses; Purple, peptide + 2 hexoses and sialic acid. (B) WT Jurkat cells, (C) Cas9 control cells, (D) XXYLT1 KO #1 (gRNA-3 transfectant), and (E) XXYLT1 KO #3 (gRNA-1 transfectant). EGF: epidermal growth factor-like HCD: higher-energy collision dissociation MS/MS: tandem mass spectrometry Glc: glucose Xyl: xylose EICs: extracted ion chromatograms WT: wild-type Cas9: CRISPR-associated protein 9 XXYLT1: xyloside α1-3xylosyltransferase 1 KO: knockout gRNA: guide RNA

Article Snippet: Total cell lysates (5 μg) were loaded and subsequently analyzed by western blotting using anti-αTubulin antibody (DSHB, 12G10, 1:4000), anti-cleaved NOTCH1 (Val1744) antibody (CST, D3B8, 1:1000), and anti-HA antibody (MBL, 561, 1:1000), and the intensity of cleaved NOTCH1 was normalized to the intensity of αTubulin.

Techniques: Modification, Tandem Mass Spectroscopy, Glycoproteomics, Control, Transfection, Mass Spectrometry, CRISPR, Knock-Out

Journal: Nagoya Journal of Medical Science

Article Title: XXYLT1 inhibits NOTCH1 activation in Jurkat cells while promoting cell proliferation

doi: 10.18999/nagjms.87.3.431

Figure Lengend Snippet: The activation of NOTCH1 is enhanced in XXYLT1 KO Jurkat clones Fig. 2A: Western Blot analysis of WT, Cas9-control, and XXYLT1 KO clones using antibodies against activated NOTCH1 (Val1744) and α-tubulin (loading control). Fig. 2B: Normalized intensities of activated NOTCH1 signals from five independent experiments. Error bars indicate standard deviation. Significance was calculated using a one-way ANOVA followed by Dunnett’s test. Fig. 2C: Western Blot analysis of NOTCH1 activation in empty vector or XXYLT1-HA -transduced WT and XXYLT1 KO cells. Fig. 2D: Normalized intensities of activated NOTCH1 signals from three independent experiments. Error bars indicate standard deviation. Significance was calculated using a one-way ANOVA followed by Tukey’s test. Fig. 2E: Flow cytometry analysis of cell surface NOTCH1 in WT, Cas9-control, and XXYLT1 KO clones. Normalized mean fluorescence intensities from four independent experiments were plotted. Error bars represent the standard deviation. Significance was calculated using a one-way ANOVA followed by Dunnett’s test. XXYLT1: xyloside α1-3xylosyltransferase 1 KO: knockout WT: wild-type Cas9: CRISPR-associated protein 9 ANOVA: analysis of variance HA: hemagglutinin tag MFI: mean fluorescence intensity

Article Snippet: Total cell lysates (5 μg) were loaded and subsequently analyzed by western blotting using anti-αTubulin antibody (DSHB, 12G10, 1:4000), anti-cleaved NOTCH1 (Val1744) antibody (CST, D3B8, 1:1000), and anti-HA antibody (MBL, 561, 1:1000), and the intensity of cleaved NOTCH1 was normalized to the intensity of αTubulin.

Techniques: Activation Assay, Clone Assay, Western Blot, Control, Standard Deviation, Plasmid Preparation, Flow Cytometry, Fluorescence, Knock-Out, CRISPR