Journal: Nature Communications

Article Title: NOTCH1 mitochondria localization during heart development promotes mitochondrial metabolism and the endothelial-to-mesenchymal transition in mice

doi: 10.1038/s41467-024-54407-7

Figure Lengend Snippet: A Representative western blot illustrating NICD1 levels in subcellular fractions from rat primary cardiac endothelial cells. Mitochondrial (Cytochrome C oxidase subunit 4, COX 4), nuclear (Histone H3, H3), and cytoplasmic (glyceraldehyde-3-phosphate dehydrogenase, GAPDH) markers were used. Three experiments were repeated independently with similar results. B Top: representative western blot illustrating NICD1 levels in whole-cell lysates and subcellular fractions from mouse primary cardiac endothelial cells and cardiomyocytes from mouse embryo hearts at E11.5 and E14.5. Bottom: Relative levels of mitochondrial NICD1 in mouse primary cardiac endothelial cells and cardiomyocytes ( n = 3). Data are presented as mean values ± SD. P values were calculated by unpaired Student’s t test with two-tailed analysis without adjustments. C Representative western blot illustrating NICD1 levels in cultured human umbilical vein endothelial cell (HUVEC), human embryonic kidney 293 cells (HEK293T), MKN45 gastric cancer cells, and BxPC-3 pancreatic cancer cells. Three experiments were repeated independently with similar results. D , E Immunofluorescence confocal microscopy images showing the colocalization of NOTCH1 (green) and mitochondria (red) in HUVEC and HEK293T cells. Results are representative of three different experiments. Scale bars: 20 μm for HUVEC and 10 μm for HEK293T. F Location and sequences of putative internal signals (M1–8) in NICD1. RAM Rbp-associated molecule domain, ANKs ankyrin repeats, TAD transcription activation domain, PEST proline (P), glutamic acid (E), serine (S), threonine (T) degradation domain, NLS nuclear localization signals. G Left: representative western blot of NICD1 levels in subcellular fractions from HUVEC and HEK293T cells overexpressing FLAG-tagged wild-type NICD1 or internal signal-deleted NICD1 (ΔM1–8). Three experiments were repeated independently with similar results. Right: Ratio of NICD1 level in mitochondria to whole-cell lysate in HUVEC and HEK293T cells. Source data are provided as a Source Data file.

Article Snippet: Cells were blocked with 5% goat serum in PBS for 30 min and stained with rabbit polyclonal antibody against NOTCH1 (1:200, #4380, CST) or rabbit polyclonal antibody against FLAG (1:100) overnight.

Techniques: Western Blot, Two Tailed Test, Cell Culture, Immunofluorescence, Confocal Microscopy, Activation Assay

Journal: Nature Communications

Article Title: NOTCH1 mitochondria localization during heart development promotes mitochondrial metabolism and the endothelial-to-mesenchymal transition in mice

doi: 10.1038/s41467-024-54407-7

Figure Lengend Snippet: A Enzymatic activity of PDH, CS, and IDH in mouse cardiac ECs isolated from mice ( n = 5). B Enzymatic activity of PDH, CS, and IDH in cells ( n = 5). C Enzymatic activity of PDH, CS, and IDH in cells transfected with siNC or NOTCH1 siRNA (si NOTCH1 ) ( n = 5). D Top: schematic of recombinant mitoNICD1 protein containing a presequence of cytochrome oxidase subunit 8 (COX8). Bottom: representative western blot illustrating intracellular distribution of wild-type NICD1 and mitoNICD1 (left) and quantification of mitochondrial NICD1 ( n = 3) (right). E Representative immunofluorescence confocal microscopy illustrating intracellular distribution of NICD1 and mitoNICD1 (left) and the quantification of mitochondrial NICD1 ( n = 3) (right) (Scale bar = 20 μm). F Representative western blot illustrating NICD1 levels (Top) and PDH activities (Bottom) in cells with different treatments ( n = 5). G Relative ratio of citrate to pyruvate in wild-type (WT) cells, NOTCH1 KO cells and NOTCH1 KO cells expressing mitoNICD1 ( n = 4). H Relative lactate level in WT cells, NOTCH1 KO cells and NOTCH1 KO cells expressing mitoNICD1 ( n = 4). I Relative ratio of citrate to pyruvate in mouse cardiac endothelial cells isolated from wild-type and Notch1 +/− mice ( n = 10). J Schematic of possible fates for [U- 13 C]glucose. K Fraction enrichment of lactate [m+3], alanine [m+3], acetyl-CoA [m+2], and citrate [m+2] in cells expressing exogenous wild-type NICD1 or not ( n = 5). L Fraction enrichment of lactate [m+3], alanine [m+3], acetyl-CoA [m+2], and citrate [m+2] in cells transfected with scramble siNC or si NOTCH1 ( n = 5). M Left: profile of oxygen consumption rate (OCR) of WT cells, NOTCH1 KO cells, and NOTCH1 KO cells expressing mitoNICD1. Oligomycin (Oligo), 2-[2-[4-(trifluoromethoxy)phenyl] hydrazinylidene]-propanedinitrile (FCCP), antimycin and rotenone (Rot/AA) were supplemented at 20, 50, and 80 min during the test. Right: Quantification of basal respiration ( n = 9) and maximal respiration indicated by OCR ( n = 6). N ATP levels of WT cells, NOTCH1 KO cells, and NOTCH1 KO cells expressing mitoNICD1 as indicated by luminescence ( n = 6). Source data are provided as a Source Data file. For A – I and K – N , data are presented as mean values ± SD; P values were calculated by unpaired Student’s t-test with two-tailed analysis without adjustments.

Article Snippet: Cells were blocked with 5% goat serum in PBS for 30 min and stained with rabbit polyclonal antibody against NOTCH1 (1:200, #4380, CST) or rabbit polyclonal antibody against FLAG (1:100) overnight.

Techniques: Activity Assay, Isolation, Transfection, Recombinant, Western Blot, Immunofluorescence, Confocal Microscopy, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: NOTCH1 mitochondria localization during heart development promotes mitochondrial metabolism and the endothelial-to-mesenchymal transition in mice

doi: 10.1038/s41467-024-54407-7

Figure Lengend Snippet: A Representative western blot illustrating the intracellular distribution of pyruvate dehydrogenase complex (PDC) components in wild-type and NOTCH1 knockout ( NOTCH1 KO) human embryonic kidney 293T (HEK293T) cells. PDHB pyruvate dehydrogenase E1 subunit beta, DLAT dihydrolipoamide S-acetyltransferase; DLD dihydrolipoyl transacetylase. B Endogenous co-immunoprecipitation (CO-IP) of NICD1 with PDC components in human umbilical vein endothelial cells (HUVEC) and HEK293T cells. C GST pull-down assay to determine in vitro binding of NICD1 to PDHA1, PDHB, DLD, and DLAT. D Representative western blot illustrating the level of each PDC component collected by endogenous immune-precipitation (IP) from wild-type HEK293T cells, NOTCH1 KO HEK293T cells, NOTCH1 KO HEK293T cells expressing exogenous NICD1, or recombinant mitochondria-targeted NICD1 (mitoNICD1). E Representative western blot illustrating the level of p-PDHA1 at serine 293 (Ser293), serine 232 (Ser232), and serine 300 (Ser300) in HUVEC cells expressing NICD1 or mitoNICD1 (left) and in HEK293T cells expressing NICD1 or mitoNICD1 (right). F Representative western blot illustrating the level of p-PDHA1 at Ser293, Ser232, and Ser300 in wild type and NOTCH1 knockdown ( NOTCH1 KD) HUVEC cells (left), and wild-type and NOTCH1 KO HEK293T cells (right). G Representative western blot illustrating the affinity of PDKs, including PDK1, PDK2, PDK3, and PDK4, and pyruvate dehydrogenase phosphatases (PDPs), including PDP1 and PDP2, to endogenous PDHA1 collected by endogenous IP from HUVEC cells expressing NICD1, mitoNICD1 or not (left), and HEK293T cells expressing NICD1, mitoNICD1, or not (right). H Representative western blot illustrating the affinity of PDK1 and PDP1 to endogenous PDHA1 collected by endogenous IP from wild-type mice and Notch1 heterozygous knockout ( Notch1 +/− ) mice. I Representative western blot illustrating the p-PDHA1 level at Ser293, Ser232, and Ser300 in mouse cardiac endothelial cells isolated from wild-type and Notch1 +/− mice. Three experiments were repeated independently with similar results for A – I . Source data are provided as a Source Data file Source data are provided as a Source Data file.

Article Snippet: Cells were blocked with 5% goat serum in PBS for 30 min and stained with rabbit polyclonal antibody against NOTCH1 (1:200, #4380, CST) or rabbit polyclonal antibody against FLAG (1:100) overnight.

Techniques: Western Blot, Knock-Out, Immunoprecipitation, Co-Immunoprecipitation Assay, Pull Down Assay, In Vitro, Binding Assay, Expressing, Recombinant, Knockdown, Isolation

Journal: Nature Communications

Article Title: NOTCH1 mitochondria localization during heart development promotes mitochondrial metabolism and the endothelial-to-mesenchymal transition in mice

doi: 10.1038/s41467-024-54407-7

Figure Lengend Snippet: A Left: representative immunofluorescence confocal microscopy showing the endothelial cell marker CD31 (red) and the mesenchymal cell marker α smooth muscle actin (α-SMA) (green) in human umbilical vein endothelial cells (HUVEC) treated with cytokines (10 mg/μL transforming growth factor β [TGF-β1] and 1 ng/μL interleukin-1β [IL-1β]), expressing exogenous NOTCH1 intracellular domain (NICD1), or mitoNICD1 in low-power field (LPF) (10×) (top, scale bar=200 μm) and high-power field (HPF) (×40) (bottom, scale bar = 50 μm)). Nuclei are stained with DAPI (blue). Right: Quantification of α-SMA positive (α-SMA + ) cells per LPF ( n = 5 biological samples per group). B Representative western blot illustrating endothelial cell markers, including CD31, vascular endothelial cadherin (VE-Cad), and vascular endothelial growth factor receptor (VEGFR), and mesenchymal cell markers, including α-SMA, N-Cadherin (N-Cad), Vimentin, and Actin, in HUVEC expressing exogenous NICD1, mitoNICD1, or not. C Representative western blot illustrating the phosphorylation of pyruvate dehydrogenase E1 subunit alpha 1 (PDHA1) (p-PDHA1) at serine 293 (Ser293) in HUVEC treated with PDH-activator dichloroacetate (DCA) (10 mmol/L), expressing mitoNICD1, or expressing mitoNICD1 and supplemented with PDH inhibitor PS-48 (100 μmol/L) as well. D Top: profile of oxygen consumption rate (OCR) of HUVEC treated with DCA (10 mmol/L), expressing mitoNICD1, or expressing mitoNICD1 and supplemented with PS-48 (100 μmol/L) as well. Bottom: Quantification of basal respiration and maximal respiration indicated by OCR ( n = 3 biological samples per group). E Left: representative immunofluorescence confocal microscopy showing CD31 (red) and α-SMA (green) in HUVEC treated with DCA (10 mmol/L), expressing mitoNICD1, or expressing mitoNICD1 and supplemented with PS-48 (100 μmol/L) as well (Scale bar=200 μm). Right: quantification of α-SMA + cells per LPF ( n = 5 biological samples per group). F Representative immunofluorescence confocal microscopy of CD31 (red) and α-SMA (green) in HUVEC treated with 5, 10, or 20 mmol/L DCA (Scale bar = 200 μm). Right: quantification of α-SMA + cells per LPF ( n = 5 biological samples per group). Source data are provided as a Source Data file. For A , D – F , data are presented as mean values ± SD; P values were calculated by unpaired Student’s t test with two-tailed analysis without adjustments.

Article Snippet: Cells were blocked with 5% goat serum in PBS for 30 min and stained with rabbit polyclonal antibody against NOTCH1 (1:200, #4380, CST) or rabbit polyclonal antibody against FLAG (1:100) overnight.

Techniques: Immunofluorescence, Confocal Microscopy, Marker, Expressing, Staining, Western Blot, Phospho-proteomics, Two Tailed Test

Journal: Nature Communications

Article Title: NOTCH1 mitochondria localization during heart development promotes mitochondrial metabolism and the endothelial-to-mesenchymal transition in mice

doi: 10.1038/s41467-024-54407-7

Figure Lengend Snippet: A Incidence of CHD in embryos of wild-type C57/Bl6 mice (WT), Notch1 +/− mice (KO), WT mice treated with low-dose homocysteine (Hcy) (0.5 g/L Hcy in drinking water orally administered to pregnant mice during E0.5 to E13.5) (WT+HCY), Notch1 +/− mice treated with Hcy (KO+HCY), WT mice treated with both Hcy and dichloroacetate (DCA) (10 mmol/L) (WT+HCY+DCA), and Notch1 +/− mice treated with both Hcy and DCA (KO+HCY+DCA). B PDH activity in mouse heart tissue isolated from WT, KO, WT+HCY, KO+HCY, WT+HCY+DCA, and KO+HCY+DCA mice ( n = 5). C Schematic of NOTCH1 protein showing the location of 15 missense mutations identified in 300 tetralogy of Fallot (TOF) patients. EGF epidermal growth factor, LNR Lin-12/Notch repeats, HD-N N-terminus of heterodimerization, HD-C C-terminus of heterodimerization, TM transmembrane domain, RAM Rbp-associated molecule domain, ANKs ankyrin repeats, TAD transcription activation domain, PEST proline, glutamic acid, serine, threonine degradation domain, NLS nuclear localization signals. D Representative western blot illustrating the level of exogenously expressed wild-type NICD1 and NICD1 mutants G1814R, A2077P, R2263Q, V2285I, and I2456V, in cytoplasm or mitochondria of human umbilical vein endothelial cells (HUVEC). Cytochrome C oxidase subunit 4 (COX 4) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were adopted as markers of mitochondria and cytoplasm fractions, respectively ( n = 3). E Co-immunoprecipitation (CO-IP) of exogenous pyruvate dehydrogenase E1 subunit beta (PDHB) and G1814R, A2077P, R2263Q, V2285I, and I2456V mutants, in HUVEC (left) and human embryonic kidney 293T (HEK293T) (right) cells ( n = 3). F Representative western blot illustrating the phosphorylation level of pyruvate dehydrogenase E1 subunit alpha 1 (p-PDHA1) at serine 293 (Ser293), serine 232 (Ser232), and serine 300 (Ser300) in wild-type and NOTCH1 knockout ( NOTCH1 KO) HEK293T cells (left) and in wild type and NOTCH1 knockdown ( NOTCH1 KD) HUVEC (right) expressing exogenous wild-type NICD1 or NICD1 mutants ( n = 3). G PDH activity detected in wild-type and NOTCH1 KO HEK293T cells (left) and in wild-type and NOTCH1 KD HUVEC (right) expressing exogenous wild-type NICD1 or NICD1 mutants ( n = 5). H Relative PDH activity detected in heart tissue isolated from mouse and TOF patients carrying NOTCH1 mutations or not (WT1, WT2, WT3, and WT4) ( n = 3). For B , D , E , G , data are presented as mean values ± SD; P values were calculated by unpaired Student’s t test with two-tailed analysis without adjustments.

Article Snippet: Cells were blocked with 5% goat serum in PBS for 30 min and stained with rabbit polyclonal antibody against NOTCH1 (1:200, #4380, CST) or rabbit polyclonal antibody against FLAG (1:100) overnight.

Techniques: Activity Assay, Isolation, Activation Assay, Western Blot, Immunoprecipitation, Co-Immunoprecipitation Assay, Phospho-proteomics, Knock-Out, Knockdown, Expressing, Two Tailed Test

Journal: Diagnostics

Article Title: NOTCH1- and CD117-Positive Stem Cells in Human Endometriosis and Adenomyosis Lesions

doi: 10.3390/diagnostics14151642

Figure Lengend Snippet: HE and IHC staining of a representative intestinal endometriosis lesion. ( A ) Intestinal endometriosis (within the ileum) with presence of cystic ectopic endometrial glands within the tunica muscularis propria; ( B ) intestinal endometriosis (purple) affecting the intestinal mucosa (left); ( C ) estrogen receptor expression in ectopic endometrial glands; ( D ) CD10 immunopositivity demarcating ectopic endometrial stroma; ( E ) NOTCH1+ cells (dark brown) in ectopic endometrial stroma (bottom) and intracystic debris; ( F ) CD117+ cells in ectopic endometrial stroma surrounding the ectopic cystic formation.

Article Snippet: The next step was incubation with 0.4% casein in phosphate-buffered saline (PBS) to reduce non-specific binding of primary antibody, followed by 1 h incubation at 37 °C with rabbit polyclonal primary antibodies against NOTCH1 (E-AB-12815, Elabscience, Houston, TX, USA), CD117 (RB-9038-RQ, Epredia, Portsmouth, NH, USA), estrogen receptor (1-ES006-07, Quartett, Berlin, Germany) and mouse monoclonal antibody against CD10 (DAK-CD10 IR 786, Dako, Nowy Sącz, Poland).

Techniques: Immunohistochemistry, Expressing

Journal: Diagnostics

Article Title: NOTCH1- and CD117-Positive Stem Cells in Human Endometriosis and Adenomyosis Lesions

doi: 10.3390/diagnostics14151642

Figure Lengend Snippet: HE and IHC staining of an adenomyosis lesion with phasic changes. ( A ) Adenomyosis lesion with secretory changes* in the glands (apical and basal vacuoles); ( B ) CD10 marker visualizing endometrial stromal cells (dark brown stain) in the ectopic lesion; ( C ) NOTCH1+ cells within the ectopic endometrial stroma; ( D ) CD117+ cells within the ectopic endometrial stroma. * Phasic changes are a rare feature of adenomyosis. In such cases, it might be preferable to refer to the lesion as myometrial endometriosis.

Article Snippet: The next step was incubation with 0.4% casein in phosphate-buffered saline (PBS) to reduce non-specific binding of primary antibody, followed by 1 h incubation at 37 °C with rabbit polyclonal primary antibodies against NOTCH1 (E-AB-12815, Elabscience, Houston, TX, USA), CD117 (RB-9038-RQ, Epredia, Portsmouth, NH, USA), estrogen receptor (1-ES006-07, Quartett, Berlin, Germany) and mouse monoclonal antibody against CD10 (DAK-CD10 IR 786, Dako, Nowy Sącz, Poland).

Techniques: Immunohistochemistry, Marker, Staining

Journal: Diagnostics

Article Title: NOTCH1- and CD117-Positive Stem Cells in Human Endometriosis and Adenomyosis Lesions

doi: 10.3390/diagnostics14151642

Figure Lengend Snippet: Box plots representing the percentage of studied stem cells in the stroma of patients with endometriosis and adenomyosis. ( A ) NOTCH1-positive cells in endometriosis ( n = 23) and adenomyosis lesions ( n = 20); ( B ) CD117-positive endometrial cells in endometriosis ( n = 23) and adenomyosis lesions ( n = 20).

Article Snippet: The next step was incubation with 0.4% casein in phosphate-buffered saline (PBS) to reduce non-specific binding of primary antibody, followed by 1 h incubation at 37 °C with rabbit polyclonal primary antibodies against NOTCH1 (E-AB-12815, Elabscience, Houston, TX, USA), CD117 (RB-9038-RQ, Epredia, Portsmouth, NH, USA), estrogen receptor (1-ES006-07, Quartett, Berlin, Germany) and mouse monoclonal antibody against CD10 (DAK-CD10 IR 786, Dako, Nowy Sącz, Poland).

Techniques:

Journal: Diagnostics

Article Title: NOTCH1- and CD117-Positive Stem Cells in Human Endometriosis and Adenomyosis Lesions

doi: 10.3390/diagnostics14151642

Figure Lengend Snippet: IHC showing NOTCH1 and CD117 expression in representative endometriosis and adenomyosis lesions. ( A ) NOTCH1-positive stromal cells (brown) in a representative tubal endometriosis lesion; ( B ) CD117-positive stromal cells (brown) in the same tubal endometriosis lesion; ( C ) spatial plot of the two types of stem cells in the same endometriotic lesion (NOTCH1+ cells—red, CD117+ cells—blue, ectopic endometrial stromal cells—green, lines—nearest neighbor between the two stem cell types); ( D ) NOTCH1-positive stromal cells (brown) in representative adenomyosis lesion; ( E ) CD117-positive stromal cells (brown) in the same adenomyosis lesion; ( F ) spatial plot of the two types of stem cells in the same adenomyosis lesion (NOTCH1+ cells—red, CD117+ cells—blue, ectopic endometrial stromal cells—green, lines—nearest neighbor between the two stem cell types).

Article Snippet: The next step was incubation with 0.4% casein in phosphate-buffered saline (PBS) to reduce non-specific binding of primary antibody, followed by 1 h incubation at 37 °C with rabbit polyclonal primary antibodies against NOTCH1 (E-AB-12815, Elabscience, Houston, TX, USA), CD117 (RB-9038-RQ, Epredia, Portsmouth, NH, USA), estrogen receptor (1-ES006-07, Quartett, Berlin, Germany) and mouse monoclonal antibody against CD10 (DAK-CD10 IR 786, Dako, Nowy Sącz, Poland).

Techniques: Expressing

Journal: Cell reports

Article Title: Akt3 activation by R-Ras in an endothelial cell enforces quiescence and barrier stability of neighboring endothelial cells via Jagged1

doi: 10.1016/j.celrep.2024.113837

Figure Lengend Snippet: (A) Western blot analysis of mock- or R-Ras38V-transduced EC lysate from confluent culture. Notch1 intercellular domain (N1ICD) was detected by an antibody specific for it. Since N3ICD-specific antibody is unavailable, it was detected by anti-Notch3 antibody based on the molecular weight of N3ICD. Notch3 [FL], full-length Notch3; Notch3 [TM], transmembrane fragment of cleaved Notch3; N3ICD, Notch3 intercellular domain. (B) Quantification of (A). N = 3 (Notch1), N = 4 (Jagged2), N = 5 (Dll4 and all Notch3 species), N = 6 (Jagged1 and N1ICD). Data are represented as the mean ± SEM. See also . (C) Immunofluorescence staining of confluent EC culture for N1ICD, N3ICD, and Jaggged1 with representative pictures and the quantification of fluorescence intensity presented as relative values (RU). DAPI nuclear staining is blue. Data are represented as the mean ± SEM. N = 3 wells. Pictures of two or three different areas were analyzed for each well. (D) Jagged1 expression in lung capillary ECs was analyzed in cdh5 -Cre; Rras f/f mice ( Rras ΔEC ) and Rras f/f wild-type control mice (WT) by immunostaining. Jagged1 intensity within the CD31 + area was quantified and normalized to the total CD31 + area. N = 3 mice. Pictures of three different areas of each lung were analyzed. Data are represented as the mean ± SEM. Scale bar, 50 μm. (E) Western blot analysis of small interfering RNA (siRNA) control (siCont) and R-Ras-silenced EC (siRRas). See also . (F) Quantification of (E). N = 3. Data are represented as the mean ± SEM. (G) Jagged1 and N1ICD levels in the control or R-Ras-silenced ECs were determined by immunofluorescence. The graphs present the fluorescence intensity as relative values (RU). DAPI nuclear staining is blue. N = 3 wells, one or two pictures were analyzed per well. Data are represented as the mean ± SEM. See also – . (H) Jagged1 was silenced (siJagged1) in the mock control or R-Ras38V-expressing ECs, and the N1ICD levels in these cells were analyzed by immunofluorescence (red). N = 2. (I) Jagged1 was silenced in the confluent culture of parental ECs, and Notch activation was analyzed by western blot. The graph presents Notch3 in relative values. N = 3. Data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, n.s., not significant.

Article Snippet: Rabbit polyclonal anti-activated Notch1 (cleaved, N1ICD) , abcam , ab8925; RRID:AB_306863.

Techniques: Western Blot, Molecular Weight, Immunofluorescence, Staining, Fluorescence, Expressing, Immunostaining, Small Interfering RNA, Activation Assay

Journal: Cell reports

Article Title: Akt3 activation by R-Ras in an endothelial cell enforces quiescence and barrier stability of neighboring endothelial cells via Jagged1

doi: 10.1016/j.celrep.2024.113837

Figure Lengend Snippet: (A) Western blot analysis of mock or R-Ras38V-expressing EC lysates from confluent culture. (B) Quantification of (A). N = 3 (p21 and p53), N = 5 (Hey1), N = 6 (Hes1). Data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, n.s., not significant. (C) RT-qPCR analyses of mRNA expression in relative values (2 −ΔCt ). N 3 or 4. Data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, n.s., not significant. See also . (D) Immunofluorescence and quantification of the relative fluorescence intensity. N = 3 wells, two or three pictures were analyzed for each well. Data are represented as the mean ± SEM. (E) Hey1 and R-Ras expression in lung capillary ECs was analyzed in cdh5 -Cre; Rras f/f mice ( Rras ΔEC ) and Rras f/f wild-type control mice (WT) by immunostaining. Hey1 intensity within the CD31 + area was quantified and normalized to the total CD31 + area. N = 3 mice; two pictures from each lung were analyzed. Zoomed-in images are 53 × 53 μm. Data are represented as the mean ± SEM. (F) Notch1 or Notch3 was silenced in mock- or R-Ras38V-transduced ECs, and the cell lysate was analyzed by western blot. N = 2. See also – . (G) The mock control (EC mock ) or R-Ras38V-transduced ECs (EC RRas38V ) were fluorescently labeled (green) and cocultured with unlabeled, non-transduced parental ECs (EC) at a 1:3 ratio. The cultures were immunostained with the indicated antibodies. The fluorescence intensity in non-transduced ECs was quantified. White circles indicate the positions of EC mock or EC RRas38V cells. N = 2 wells, three or four pictures were analyzed for each well. Data are represented as the mean ± SEM.

Article Snippet: Rabbit polyclonal anti-activated Notch1 (cleaved, N1ICD) , abcam , ab8925; RRID:AB_306863.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Immunofluorescence, Fluorescence, Immunostaining, Labeling

Journal: Cell reports

Article Title: Akt3 activation by R-Ras in an endothelial cell enforces quiescence and barrier stability of neighboring endothelial cells via Jagged1

doi: 10.1016/j.celrep.2024.113837

Figure Lengend Snippet: (A) The gene expression levels in HUVECs were compared between Akt isoforms by RNA-seq. N = 3. Data are represented as the mean ± SD. (B and C) The protein level of each Akt isoform (N = 3–5) and Ser473 phosphorylation of all Akts were determined in R-Ras38V-transduced (B) or R-Ras-silenced (C) ECs by western blot. For quantification, pAkt Ser473 was normalized to the total Akt. Data are represented as the mean ± SEM. (D) Phosphorylation of each Akt isoform was determined in mock- or R-Ras38V-transduced ECs by immunoprecipitation of the total pAkt Ser473 followed by western blot of each isoform. (E) Each Akt isoform was silenced in mock- or R-Ras38V-transduced ECs and the protein levels of Jagged1 and p21 were determined in these cells. (F) The Akt3 isoform was silenced in R-Ras38V-transduced ECs, and Notch1 and Notch3 activation as well as the protein levels of Jagged1, p21, VE-cadherin, and UNC5b were determined. Data are represented as the mean ± SEM. (G) Akt3 was silenced in mock- (EC mock ) or R-Ras38V-transduced ECs (EC RRas38V ) that were cocultured with parental ECs (EC), and Jagged1 expression was evaluated. *p < 0.05, **p < 0.01; n.s., not significant.

Article Snippet: Rabbit polyclonal anti-activated Notch1 (cleaved, N1ICD) , abcam , ab8925; RRID:AB_306863.

Techniques: Expressing, RNA Sequencing Assay, Western Blot, Immunoprecipitation, Activation Assay

Journal: Cell reports

Article Title: Akt3 activation by R-Ras in an endothelial cell enforces quiescence and barrier stability of neighboring endothelial cells via Jagged1

doi: 10.1016/j.celrep.2024.113837

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-activated Notch1 (cleaved, N1ICD) , abcam , ab8925; RRID:AB_306863.

Techniques: Virus, Expressing, Plasmid Preparation, Recombinant, Isolation, MTT Assay, Western Blot, Software