Journal: American journal of physiology. Heart and circulatory physiology

Article Title: Early experimental hypertension preserves the myocardial microvasculature but aggravates cardiac injury distal to chronic coronary artery obstruction.

doi: 10.1152/ajpheart.00516.2010

Figure Lengend Snippet: Fig. 4. A: expression of myocardial collagen I, matrix metalloproteinase (MMP)-9, zonula occludens-1 (ZO-1), vascular endothelial growth factor (VEGF), basic fibroblastic growth factor (bFGF), VEGF receptor FLK-1, Notch-1, and its receptor Delta-like receptor 4 (DLL-4). B: respective quantitation showing increased fibrosis and angiogenic factors in CAS and in CASHT compared with normal and increased bFGF and Notch-1 only in CASHT. *P 0.05 vs. normal; †P 0.05 vs. HT.

Article Snippet: The primary antibodies (rabbit IgG) used for blotting were anti-ZO-1 (1:1,000, Zymed), collagen I (1:1,000, Cosmo Bio), active and proMMP-9 (1:500, Millipore), notch1 (1:500, Novus), DLL4 (1:500, Novus), VEGF (1:200, Santa Cruz), FLK-1 (1:200, Santa Cruz), and bFGF (1:500, Millipore).

Techniques: Expressing, Quantitation Assay

Journal: Scientific Reports

Article Title: Notch1 regulates Orai1 and Orai3 expression in breast cancer cells

doi: 10.1038/s41598-025-33071-x

Figure Lengend Snippet: Effect of Notch1 intracellular domain (N1ICD) expression on the expression of total Orai1 and Orai1α and Orai1β isoforms in non-tumoral breast epithelial cells and breast cancer cells. ( A – E ) Non-tumoral breast epithelial MCF10A cells ( A ), TNBC MDA-MB-231 ( B ) and BT20 cells ( C ), as well as luminal breast cancer MCF7 ( D ) and T47D cells ( E ) were transfected with myc-N1ICD fragment (myc-N1ICD) or empty vector (Control) and 24 h later cells were lysed. Cell lysates were subjected to 10% SDS-PAGE and Western blotting with anti-myc or anti-Orai1 antibody, as described in Materials and Methods. Membranes were reprobed with anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. n = 10, 13,10, 7 and 11 separate experiments for MCF10A, MDA-MB-231, BT20, MCF-7 and T47D, respectively. ( F – H ) Bar graphs represent the quantification of Orai1 ( F ), Orai1α ( G ) or Orai1β ( H ) protein expression as fold change over the level in mock-transfected cells (Control) and presented as means ± S.E.M. Data were statistically analyzed using Mann–Whitney U test. * p < 0.05 and ** p < 0.01.

Article Snippet: Immunodetection of Orai1, Orai3, Notch1, myc-tagged proteins, flag-tagged proteins, and β-actin was performed by incubating the membranes with primary antibodies diluted in EveryBlot Blocking Buffer for 1 h at room temperature, with the following dilutions: 1–500 for anti-Notch1 antibody (rabbit polyclonal Notch1 antibody, catalog number: 100–401−407, Rockland Immunochemicals, Inc, Pottstown, PA, USA), anti-myc antibody (mouse monoclonal anti-c-Myc antibody (Clone 9E10; RRID: AB_2533008; catalog number 13–2500, ThermoFisher Scientific) and anti-flag antibody (mouse monoclonal anti-DYKDDDDK (flag) antibody (Clone FG4R; RRID: AB_2537626; catalog number MA1-91878-HRP, ThermoFisher Scientific).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, SDS Page, Western Blot, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Notch1 regulates Orai1 and Orai3 expression in breast cancer cells

doi: 10.1038/s41598-025-33071-x

Figure Lengend Snippet: Effect of Notch1 intracellular domain (N1ICD) expression on the expression of STIM1 and Orai3 in non-tumoral breast epithelial cells and breast cancer cells. ( A – E ) Non-tumoral breast epithelial MCF10A cells ( A ), TNBC MDA-MB-231 ( B ) and BT20 cells ( C ), as well as luminal breast cancer MCF7 ( D ) and T47D cells ( E ) were transfected with myc-N1ICD fragment (myc-N1ICD) or empty vector (Control) and 24 h later cells were lysed. Cell lysates were subjected to 10% SDS-PAGE and Western blotting with anti-myc, anti-STIM1 or anti-Orai3 antibody, as described in Materials and Methods. Membranes were reprobed with anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. * Non-specific band. n = 9, 13, 8, 11 and 10 separate experiments for Orai3 and 4, 6, 4, 3 and 5 separate experiments for STIM1 in MCF10A, MDA-MB-231, BT20, MCF-7 and T47D, respectively. ( F , G ) Bar graphs represent the quantification of Orai3 ( F ) or STIM1 ( G ) protein expression as fold change over the level in mock-transfected cells (Control) and presented as means ± S.E.M. Data were statistically analyzed using Mann–Whitney U test. * p < 0.05 and ** p < 0.01.

Article Snippet: Immunodetection of Orai1, Orai3, Notch1, myc-tagged proteins, flag-tagged proteins, and β-actin was performed by incubating the membranes with primary antibodies diluted in EveryBlot Blocking Buffer for 1 h at room temperature, with the following dilutions: 1–500 for anti-Notch1 antibody (rabbit polyclonal Notch1 antibody, catalog number: 100–401−407, Rockland Immunochemicals, Inc, Pottstown, PA, USA), anti-myc antibody (mouse monoclonal anti-c-Myc antibody (Clone 9E10; RRID: AB_2533008; catalog number 13–2500, ThermoFisher Scientific) and anti-flag antibody (mouse monoclonal anti-DYKDDDDK (flag) antibody (Clone FG4R; RRID: AB_2537626; catalog number MA1-91878-HRP, ThermoFisher Scientific).

Techniques: Expressing, Transfection, Plasmid Preparation, Control, SDS Page, Western Blot, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Notch1 regulates Orai1 and Orai3 expression in breast cancer cells

doi: 10.1038/s41598-025-33071-x

Figure Lengend Snippet: Effect of Jagged-1 in the expression of Orai1 and Orai3 in non-tumoral breast epithelial cells and breast cancer cells. ( A – E ) Non-tumoral breast epithelial MCF10A cells ( A ), TNBC MDA-MB-231 ( B ) and BT20 cells ( C ), as well as luminal breast cancer MCF7 ( D ) and T47D cells ( E ) were stimulated with 50 µM Jagged-1 (Jag-1) or the vehicle (Mock) for 48 h and lysed. Cell lysates were subjected to 10% SDS-PAGE and Western blotting with the anti-Notch1, anti-Orai1 or anti-Orai3 antibody, as described in Materials and Methods. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. n = 5–9 separate experiments. ( F – H ) Bar graphs represent the quantification of protein expression as fold change over the level in vehicle-transfected cells and presented as means ± S.E.M. Data were statistically analyzed using Mann–Whitney U test. * p < 0.05 and ** p < 0.01.

Article Snippet: Immunodetection of Orai1, Orai3, Notch1, myc-tagged proteins, flag-tagged proteins, and β-actin was performed by incubating the membranes with primary antibodies diluted in EveryBlot Blocking Buffer for 1 h at room temperature, with the following dilutions: 1–500 for anti-Notch1 antibody (rabbit polyclonal Notch1 antibody, catalog number: 100–401−407, Rockland Immunochemicals, Inc, Pottstown, PA, USA), anti-myc antibody (mouse monoclonal anti-c-Myc antibody (Clone 9E10; RRID: AB_2533008; catalog number 13–2500, ThermoFisher Scientific) and anti-flag antibody (mouse monoclonal anti-DYKDDDDK (flag) antibody (Clone FG4R; RRID: AB_2537626; catalog number MA1-91878-HRP, ThermoFisher Scientific).

Techniques: Expressing, SDS Page, Western Blot, Control, Transfection, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Notch1 regulates Orai1 and Orai3 expression in breast cancer cells

doi: 10.1038/s41598-025-33071-x

Figure Lengend Snippet: Effect of DAPT in the expression of Orai1 and Orai3 in non-tumoral breast epithelial cells and breast cancer cells. ( A – E ) Non-tumoral breast epithelial MCF10A cells ( A ), TNBC MDA-MB-231 ( B ) and BT20 cells ( C ), as well as luminal breast cancer MCF7 ( D ) and T47D cells ( E ) were treated with 20 µM DAPT or the vehicle (DMSO) for 24, 48–72 h and lysed. Cell lysates were subjected to 10% SDS-PAGE and Western blotting with the anti-Notch1, anti-Orai1 or anti-Orai3 antibody, as described in Materials and Methods. Membranes were reprobed with the anti-β-actin antibody for protein loading control. Molecular masses indicated on the right were determined using molecular-mass markers run in the same gel. n = 4–6 separate experiments. Bar graphs represent the quantification of protein expression as fold change over the level in vehicle-treated cells and presented as means ± S.E.M. Data were statistically analyzed using Mann–Whitney U test. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: Immunodetection of Orai1, Orai3, Notch1, myc-tagged proteins, flag-tagged proteins, and β-actin was performed by incubating the membranes with primary antibodies diluted in EveryBlot Blocking Buffer for 1 h at room temperature, with the following dilutions: 1–500 for anti-Notch1 antibody (rabbit polyclonal Notch1 antibody, catalog number: 100–401−407, Rockland Immunochemicals, Inc, Pottstown, PA, USA), anti-myc antibody (mouse monoclonal anti-c-Myc antibody (Clone 9E10; RRID: AB_2533008; catalog number 13–2500, ThermoFisher Scientific) and anti-flag antibody (mouse monoclonal anti-DYKDDDDK (flag) antibody (Clone FG4R; RRID: AB_2537626; catalog number MA1-91878-HRP, ThermoFisher Scientific).

Techniques: Expressing, SDS Page, Western Blot, Control, MANN-WHITNEY

Journal: Journal of Cancer Research and Therapeutics

Article Title: Specific inhibition of Notch1 signaling suppresses properties of lung cancer stem cells

doi: 10.4103/jcrt.jcrt_482_17

Figure Lengend Snippet: Figure 1: Isolation and identification of CD44+/CD24− cells from A549 cells and the detection of Notch1 expression. (a) Spheres formed by isolated CD44+/CD24− cells under serum‑free culture conditions. (b) Expression of cancer stem cells marker ALDH1A in CD44+/CD24− cells and unsorted A549 cells was detected by immunocytochemistry. (c) Expression of Notch1 mRNA in CD44+/CD24− cells and unsorted A549 cells was detected by polymerase chain reaction. (d) Expression of Notch1 protein in CD44+/CD24− cells and unsorted A549 cells was detected by western blot

Article Snippet: Inhibition of Notch1 by DAPT and small interfering RNA Isolated CD44+/CD24− cells were treated with γ‐secretase inhibitor ‐ DAPT in different concentrations (0, 25, 50, and 75 μM) for 48 h to block the Notch1 signaling pathway. siRNAs were also constructed to block Notch1. siRNA vectors targeting to Notch1 gene were synthesized by Santa Cruz Biotechnology.

Techniques: Isolation, Expressing, Marker, Immunocytochemistry, Polymerase Chain Reaction, Western Blot

Journal: Journal of Cancer Research and Therapeutics

Article Title: Specific inhibition of Notch1 signaling suppresses properties of lung cancer stem cells

doi: 10.4103/jcrt.jcrt_482_17

Figure Lengend Snippet: Figure 3: Small interfering RNAs targeting Notch1 were constructed and the interfering efficiency was detected. (a) The expression of Notch1 mRNA in CD44+/CD24− cells treated with different siRNAs was detected by polymerase chain reaction. (b) The expression of Notch1 protein in CD44+/CD24− cells treated with different siRNAs was detected by western blot

Article Snippet: Inhibition of Notch1 by DAPT and small interfering RNA Isolated CD44+/CD24− cells were treated with γ‐secretase inhibitor ‐ DAPT in different concentrations (0, 25, 50, and 75 μM) for 48 h to block the Notch1 signaling pathway. siRNAs were also constructed to block Notch1. siRNA vectors targeting to Notch1 gene were synthesized by Santa Cruz Biotechnology.

Techniques: Construct, Expressing, Polymerase Chain Reaction, Western Blot

Journal: Journal of Cancer Research and Therapeutics

Article Title: Specific inhibition of Notch1 signaling suppresses properties of lung cancer stem cells

doi: 10.4103/jcrt.jcrt_482_17

Figure Lengend Snippet: Figure 2: CD44+/CD24− cells were treated with Notch1 inhibitor DAPT. (a) Expression of Notch1 mRNA in CD44+/CD24− cells treated with DAPT in different concentrations (0, 25, 50, and 75 μM) for 72 h was detected by polymerase chain reaction. (b) Expression of Notch1 protein in CD44+/CD24− cells treated with DAPT in different concentrations (0, 25, 50, and 75 μM) for 72 h was detected by western blot. (c) Proliferation of CD44+/CD24− cells after treatment with 50 μM DAPT for 2 weeks was detected by colony‑forming assay

Article Snippet: Inhibition of Notch1 by DAPT and small interfering RNA Isolated CD44+/CD24− cells were treated with γ‐secretase inhibitor ‐ DAPT in different concentrations (0, 25, 50, and 75 μM) for 48 h to block the Notch1 signaling pathway. siRNAs were also constructed to block Notch1. siRNA vectors targeting to Notch1 gene were synthesized by Santa Cruz Biotechnology.

Techniques: Expressing, Polymerase Chain Reaction, Western Blot

Journal: Journal of Cancer Research and Therapeutics

Article Title: Specific inhibition of Notch1 signaling suppresses properties of lung cancer stem cells

doi: 10.4103/jcrt.jcrt_482_17

Figure Lengend Snippet: Figure 4: CD44+/CD24− cells were treated with Notch1 siRNA-2. (a) The protein expressions of Notch1 and ALDH1A in CD44+/CD24− cells treated with siRNA‑4 and control siRNAs were analyzed by western blot (left) and gray scale scanning (right). (b) Spheres formed by CD44+/CD24− cells treated with siRNA‑4 and control siRNAs. (c) Quantitative analysis of sphere‑forming efficiency (left) and sphere numbers (right) formed from CD44+/CD24− cells treated with siRNA‑4 and control siRNAs. (d) Cell proliferation of CD44+/CD24− cells treated with different siRNAs was detected by CCK‑8

Article Snippet: Inhibition of Notch1 by DAPT and small interfering RNA Isolated CD44+/CD24− cells were treated with γ‐secretase inhibitor ‐ DAPT in different concentrations (0, 25, 50, and 75 μM) for 48 h to block the Notch1 signaling pathway. siRNAs were also constructed to block Notch1. siRNA vectors targeting to Notch1 gene were synthesized by Santa Cruz Biotechnology.

Techniques: Control, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Indoxyl Sulfate Inhibits Osteogenesis in Bone Marrow Mesenchymal Stem Cells through the AhR/Hes1 Pathway

doi: 10.3390/ijms25168770

Figure Lengend Snippet: IS upregulated the expression of hairy/enhancer of split-1 ( Hes1 ) through AhR. ( A ) qRT-PCR revealed the mRNA levels of Notch receptors and Hes1 in D1 cells treated with IS for at least 4 and 8 h or left untreated. ( B ) Western blotting revealed the protein levels of cleaved-Notch1 and Hes1 in D1 cells treated with IS or a vehicle control for 1 and 2 days. ( C ) qRT-PCR indicated the level of Hes1 expression in control or IS-treated D1 cells subjected to AhR antagonist (CH-223191) treatment (upper panel) or Ahr knockdown (KD) (lower panel) for 4 and 8 h. Data are presented as mean ± standard deviation values ( n = 3; * p < 0.05, ** p < 0.01, *** p < 0.001; * compared with the control. # p < 0.05, ## p < 0.01, ### p < 0.001; # compared with the IS-treated group; Student t -test ( A , B ) and analysis of variance ( C )).

Article Snippet: The membrane was blocked with 5% skim milk–Tris-buffered saline with 0.1% Tween 20 for 1 h. After washing, the membrane was incubated overnight at 4 °C with primary antibodies against Runx2 (Abcam, Cambridge, UK, Cat# ab23981), Bmp2 (Abcam, Cat# ab14933), Alp (Abcam, Cat# ab203106), Oc (Abcam, Cat# ab93876), AhR (Santa Cruz Biotechnology, Inc., Dallas, TX, USA, Cat# sc-133088), Hes1 (NOVUS Biologicals, Centennial, CO, USA, Cat# NBP1-47791), and cleaved-Notch1 (Elabscience Biotechnology Co., Ltd., Wuhan, China, Cat# E-AB-30054).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control, Knockdown, Standard Deviation