Journal: OncoTargets and therapy

Article Title: Epigallocatechin gallate inhibits the proliferation of colorectal cancer cells by regulating Notch signaling

doi: 10.2147/OTT.S40914

Figure Lengend Snippet: The effect of EGCG on expressions of Notch1, Notch2, HES1 , and JAG1 in the colorectal cancer cells: ( A ) the expression of HES1 ; ( B ) the expression of JAG1 ; ( C ) the expression of Notch1 ; and ( D ) the expression of Notch2 . Notes: The x-axis shows the Lovo, SW480, HT-29, and HCT-8 colorectal cell lines. The y-axis shows the mRNA of the colorectal cell lines after treatment with EGCG. An independent-sample t -test was used to analyze the mRNA levels of the colorectal cell lines. Abbreviation: EGCG, epigallocatechin gallate.

Article Snippet: The primary antibodies used were Abcam Hes1 antibody (ab71559), CST Jagged1 (28H8Rabbit mAb), CST Notch1 (D1E11 XP™ Rabbit mAb), and CST Notch2 (8A1Rabbit mAb) (Sigma-Aldrich, St Louis, MO, USA).

Techniques: Expressing

Journal: Journal of translational medicine

Article Title: MicroRNA-143 acts as a tumor suppressor through Musashi-2/DLL1/Notch1 and Musashi-2/Snail1/MMPs axes in acute myeloid leukemia.

doi: 10.1186/s12967-023-04106-6

Figure Lengend Snippet: Fig. 5 MSI2 activity is negatively modulated by miR-143 in vitro (A) Key factors of Notch1 signaling pathway were determined in HEL and HL-60 cells transfected with miR-143 or miR-NC mimic. (B, C) Key factors of Notch1 signaling pathway (B) and tumor metastasis-associated proteins (C) were measured in Lenti- MiR-143 vector- or Lenti-Control vector-transfecting HEL cells. (D) Cell growth of MSI2-OE. HEL and NC. HEL cells transfected with miR-143 or miR-NC mimic was analyzed by CCK-8 assay. (E) Clonogenic capacity of Lenti-MSI2 vector- or Lenti-Control vector-transfecting HEL cells after the transfection with miR-143 or miR-NC mimic. Representative pictures (above) and statistical analysis diagram (below) were illustrated, scale bar = 200 μm. (F) Apoptosis in Lenti-MSI2 vector- or Lenti-Control vector-transfecting HEL cells transfected with miR-143 or miR-NC mimic. (G, H) Expression of MSI2, key factors of Notch1 signaling pathway and cancer stemness-related proteins (G) and tumor metastasis-associated proteins (H) were shown in Lenti-MSI2 vector- or Lenti-Control vector-transfecting HEL cells transfected with miR-143 or miR-NC mimic. Data are expressed as mean ± SD (error bars). * P < 0.05, ** P < 0.01 and ***P < 0.001, t-test

Article Snippet: Then the protein was determined using antibodies against MSI2 (Clone: EP1305Y, Abcam, Cambridge, UK), DLL1 (Cat#202301-AP, Proteintech, Wuhan, CN), HES1 (Clone: D6P2U, Cell Signaling Technology (CST), Beverly, MA, USA), cleaved NOTCH1 (Clone: (D1E11) XP®, CST), NANOG (Cat#A3232, ABclonal, Wuhan, CN), OCT4 (Cat#A7920, ABclonal), SOX2 (Cat#A0561, ABclonal), SNAIL1 (Clone: C15D3, CST), MMP2 (Clone: D4M2N, CST), MMP9 (Clone: (D6O3H) XP®, CST), β-actin (Cat#AF7018, Effinity) and GAPDH (Cat#60004-1-1 g, Proteintech), at 1/1,000 dilution for incubation overnight at 4 °C, respectively.

Techniques: Activity Assay, In Vitro, Transfection, Plasmid Preparation, Control, CCK-8 Assay, Expressing

Journal: Journal of translational medicine

Article Title: MicroRNA-143 acts as a tumor suppressor through Musashi-2/DLL1/Notch1 and Musashi-2/Snail1/MMPs axes in acute myeloid leukemia.

doi: 10.1186/s12967-023-04106-6

Figure Lengend Snippet: Fig. 7 Schematic illustrates the mechanism by which miR-143 directly binds to MSI2 and inhibits its efficacy in AML progression. MSI2/DLL1/ Notch1 axis: MSI2 binds directly to DLL1 mRNA and stabilize it at the post-transcriptional level. Then DLL1 agonistic ligands trigger proteolytic cleavage of Notch1 receptors to generate the NICD. NICD/CSL-dependent transcriptional activation of target gene HES1 is upregulated by canonical Notch signaling cascades to maintain stemness of AML cells. MSI2/Snail1/MMPs axis: MSI2 stabilizes Snail1 mRNA and induced transcriptional upregulation of Snail1, which in turn activated both MMP2 and MMP9 to promote AML cell migration. AML, Acute myeloid leukemia; MiR-143, MicroRNA-143; MSI2, Musashi-2; DLL, delta-like canonical Notch ligand; NICD, Notch intracellular domain; MAML, mastermind like protein; CSL, cBF1-suppressor of hairless-LAG1; HES1, hes family bHLH transcription factor 1; Snail1: snail family transcriptional repressor 1; MMP matrix metalloproteinase

Article Snippet: Then the protein was determined using antibodies against MSI2 (Clone: EP1305Y, Abcam, Cambridge, UK), DLL1 (Cat#202301-AP, Proteintech, Wuhan, CN), HES1 (Clone: D6P2U, Cell Signaling Technology (CST), Beverly, MA, USA), cleaved NOTCH1 (Clone: (D1E11) XP®, CST), NANOG (Cat#A3232, ABclonal, Wuhan, CN), OCT4 (Cat#A7920, ABclonal), SOX2 (Cat#A0561, ABclonal), SNAIL1 (Clone: C15D3, CST), MMP2 (Clone: D4M2N, CST), MMP9 (Clone: (D6O3H) XP®, CST), β-actin (Cat#AF7018, Effinity) and GAPDH (Cat#60004-1-1 g, Proteintech), at 1/1,000 dilution for incubation overnight at 4 °C, respectively.

Techniques: Activation Assay, Migration

Journal: Cell Death & Disease

Article Title: Detrimental effects of Notch1 signaling activated by cadmium in renal proximal tubular epithelial cells

doi: 10.1038/cddis.2014.339

Figure Lengend Snippet: Involvement of Notch1 signaling in CdCl 2 -induced cellular damage in HK-2 cells. ( a and b ) Cells were incubated with 20 μ M CdCl 2 (Cd) for the indicated time. The untreated control is labeled 0 h. Cell lysates were subjected to western blotting using antibodies against Notch1-NICD, Nohch1-NTM, and actin ( a ). Equal amounts of protein (20 μ g) in nuclear and cytoplasmic extracts were subjected to western blotting using antibodies against Notch1-NICD, MEK1/2, lamin A/C, and actin. MEK1 and lamin A/C served as a loading control for cytoplasmic and nuclear extracts, respectively ( b ). ( c – e ) Cells transfected with control siRNA, Notch1 siRNA-1, or Notch1 siRNA-2 were incubated with or without 20 μ M CdCl 2 (Cd) for 12 h ( c ), 24 h ( d ), or 30 h ( e ). Cell lysates were subjected to western blotting using antibodies against Notch1-NICD, Notch1-NTM, and actin ( c ). Phase-contrast micrographs were taken ( d ). The viability of cells was determined by trypan blue exclusion assay. Each value is the percentage of trypan blue-positive cells and reflects the mean±S.D. of three experiments with duplicate assays in each experiment. * P <0.05, ** P <0.01 versus CdCl 2 -treated cells transfected with control siRNA ( e ). ( f – h ) Cells were incubated with 0.1% DMSO or 40 μ M DAPT for 1 h and then incubated with or without 20 μ M CdCl 2 (Cd) for 12 h ( f ) or 30 h ( g and h ). Cell lysates were subjected to western blotting using antibodies against Notch1-NICD, Notch1-NTM, and actin ( f ). Phase-contrast micrographs were taken ( g ). The viability of cells was determined by trypan blue exclusion assay. Each value is the percentage of trypan blue-positive cells and reflects the mean±S.D. of three experiments with duplicate assays in each experiment. ** P <0.01 versus CdCl 2 -treated cells incubated with DMSO ( h ). Immunoblots shown are representative of at least three independent experiments

Article Snippet: Antibodies against phospho-EGFR (Tyr1068), total EGFR (D38B1) XP, phospho-Akt (Thr308) (C31E5E), total Akt (pan) (C67E7), phospho-p70 S6 kinase (Thr389) (108D2), phospho-GSK-3 α / β (Ser21/9) (37F11), total GSK3- α / β (D75D3) XP, cleaved Notch1 (Val1744) (D3B8), Notch1 (D1E11) XP, Jagged1 (28H8), Jagged2 (C23D2), Snail (C15D3), E-cadherin (24E10), phospho-p53 (Ser15), and MEK1/2 were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). p53 (DO-1), Notch1 (C-20), actin (I-19), and lamin A/C (14/LaminAC) antibodies were obtained from Santa Cruz Biotechnology.

Techniques: Incubation, Control, Labeling, Western Blot, Transfection, Trypan Blue Exclusion Assay

Journal: Cell Death & Disease

Article Title: Detrimental effects of Notch1 signaling activated by cadmium in renal proximal tubular epithelial cells

doi: 10.1038/cddis.2014.339

Figure Lengend Snippet: Jagged1 and Jagged2 are involved in CdCl 2 -induced cellular damage in HK-2 cells. ( a ) Cells were incubated with or without 20 μ M CdCl 2 (Cd) for the indicated time. Cell lysates were subjected to western blotting using antibodies against Jagged1, Jagged2, and actin. ( b – d ) Cells transfected with control siRNA, Jagged1 siRNA, or Jagged2 siRNA were incubated with or without 20 μ M CdCl 2 (Cd) for 12 h ( b ), 12 h ( c ), or 30 h ( d ). Cell lysates were subjected to western blotting using antibodies against Notch1-NICD, Jagged1 (left), Jagged2 (right), and actin ( b ). Phase-contrast micrographs were taken ( c ). The viability of cells was determined by trypan blue exclusion assay. Each value is the percentage of trypan blue-positive cells and reflects the mean±S.D. of three experiments with duplicate assays in each experiment. * P <0.05 versus CdCl 2 -treated cells transfected with control siRNA ( d ). Immunoblots shown are representative of at least three independent experiments

Article Snippet: Antibodies against phospho-EGFR (Tyr1068), total EGFR (D38B1) XP, phospho-Akt (Thr308) (C31E5E), total Akt (pan) (C67E7), phospho-p70 S6 kinase (Thr389) (108D2), phospho-GSK-3 α / β (Ser21/9) (37F11), total GSK3- α / β (D75D3) XP, cleaved Notch1 (Val1744) (D3B8), Notch1 (D1E11) XP, Jagged1 (28H8), Jagged2 (C23D2), Snail (C15D3), E-cadherin (24E10), phospho-p53 (Ser15), and MEK1/2 were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). p53 (DO-1), Notch1 (C-20), actin (I-19), and lamin A/C (14/LaminAC) antibodies were obtained from Santa Cruz Biotechnology.

Techniques: Incubation, Western Blot, Transfection, Control, Trypan Blue Exclusion Assay

Journal: Cell Death & Disease

Article Title: Detrimental effects of Notch1 signaling activated by cadmium in renal proximal tubular epithelial cells

doi: 10.1038/cddis.2014.339

Figure Lengend Snippet: Modulation of Notch1 signaling by p53 in HK-2 cells exposed to CdCl 2 . ( a and b ) Cells were incubated with 20 μ M CdCl 2 (Cd) for the indicated time. The untreated control is labeled 0 h. Cell lysates were subjected to western blotting using antibodies against p53, phopho-p53, and actin ( a ). Equal amounts of protein (20 μ g) in nuclear and cytoplasmic extracts were subjected to western blotting using antibodies against p53, MEK1/2, lamin A/C, and actin. MEK1 and lamin A/C served as a loading control for cytoplasmic and nuclear extracts, respectively. Data are from the same experiment shown in ( b ). ( c – e ) Cells were incubated with 0.1% DMSO or 40 μ M pifithrin- α for 1 h and then incubated with or without 20 μ M CdCl 2 (Cd) for 24 h ( c ), 30 h ( d ), or 12 h ( e ). Phase-contrast micrographs were taken ( c ). The viability of cells was determined by trypan blue exclusion assay. Each value is the percentage of trypan blue-positive cells and reflects the mean±S.D. of three experiments with duplicate assays in each experiment. ** P <0.01 versus CdCl 2 -treated cells incubated with DMSO ( d ). Cell lysates were subjected to western blotting using antibodies against Notch1-NICD, Notch1-NTM, and actin ( e ). ( f ) Cells transfected with control siRNA or Notch1 siRNA-1 were incubated with or without 20 μ M CdCl 2 (Cd) for 12 h. Cell lysates were subjected to western blotting using antibodies against p53, phospho-p53, Notch1-NICD, and actin. Immunoblots shown are representative of at least three independent experiments

Article Snippet: Antibodies against phospho-EGFR (Tyr1068), total EGFR (D38B1) XP, phospho-Akt (Thr308) (C31E5E), total Akt (pan) (C67E7), phospho-p70 S6 kinase (Thr389) (108D2), phospho-GSK-3 α / β (Ser21/9) (37F11), total GSK3- α / β (D75D3) XP, cleaved Notch1 (Val1744) (D3B8), Notch1 (D1E11) XP, Jagged1 (28H8), Jagged2 (C23D2), Snail (C15D3), E-cadherin (24E10), phospho-p53 (Ser15), and MEK1/2 were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). p53 (DO-1), Notch1 (C-20), actin (I-19), and lamin A/C (14/LaminAC) antibodies were obtained from Santa Cruz Biotechnology.

Techniques: Incubation, Control, Labeling, Western Blot, Trypan Blue Exclusion Assay, Transfection

Journal: Cell Death & Disease

Article Title: Detrimental effects of Notch1 signaling activated by cadmium in renal proximal tubular epithelial cells

doi: 10.1038/cddis.2014.339

Figure Lengend Snippet: Cross-talk between the Notch1 and PI3K/Akt signaling pathways in HK-2 cells exposed to CdCl 2 . ( a ) Cells were incubated with 20 μ M CdCl 2 (Cd) for the indicated time. The untreated control is labeled 0 h. Cell lysates were subjected to western blotting using antibodies against phospho-EGFR, total EGFR, phospho-Akt, total Akt, phospho-GSK-3 α , phospho-GSK-3 β , total GSK-3 α / β , phospho-S6K, and actin. ( b – e ) Cells were incubated with 0.1% DMSO, 25 μ M LY294002 ( b and d ), or 10 μ M MK2206 ( c and e ) for 1 h and then incubated with or without 20 μ M CdCl 2 (Cd) for 24 h ( b and c ) or 30 h ( d and e ). Phase-contrast micrographs were taken ( b and c ). The viability of cells was determined by trypan blue exclusion assay. Each value is the percentage of trypan blue-positive cells and reflects the mean±S.D. of three experiments with duplicate assays in each experiment. ** P <0.01 versus CdCl 2 -treated cells incubated with DMSO ( d and e ). ( f ) Cells transfected with control siRNA or Notch1 siRNA-1 were incubated with or without 20 μ M CdCl 2 (Cd) for 12 h. Cell lysates were subjected to western blotting using antibodies against phospho-EGFR, total EGFR, phospho-Akt, total Akt, phospho-S6K, Notch1-NICD, and actin. Data are from the same experiment shown in . ( g ) Cells were incubated with 0.1% DMSO, 2.5 μ M AG1478, 5 μ M PPP, or 25 μ M LY294002 for 1 h and then incubated with or without 20 μ M CdCl 2 (Cd) for 12 h. Cell lysates were subjected to western blotting using antibodies against Notch1-NICD, phospho-Akt, total Akt, and actin. Immunoblots shown are representative of at least three independent experiments

Article Snippet: Antibodies against phospho-EGFR (Tyr1068), total EGFR (D38B1) XP, phospho-Akt (Thr308) (C31E5E), total Akt (pan) (C67E7), phospho-p70 S6 kinase (Thr389) (108D2), phospho-GSK-3 α / β (Ser21/9) (37F11), total GSK3- α / β (D75D3) XP, cleaved Notch1 (Val1744) (D3B8), Notch1 (D1E11) XP, Jagged1 (28H8), Jagged2 (C23D2), Snail (C15D3), E-cadherin (24E10), phospho-p53 (Ser15), and MEK1/2 were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). p53 (DO-1), Notch1 (C-20), actin (I-19), and lamin A/C (14/LaminAC) antibodies were obtained from Santa Cruz Biotechnology.

Techniques: Protein-Protein interactions, Incubation, Control, Labeling, Western Blot, Trypan Blue Exclusion Assay, Transfection

Journal: Cell Death & Disease

Article Title: Detrimental effects of Notch1 signaling activated by cadmium in renal proximal tubular epithelial cells

doi: 10.1038/cddis.2014.339

Figure Lengend Snippet: The Notch1 and PI3K/Akt signaling pathways are involved in the regulation of Snail and E-cadherin expression in HK-2 cells exposed to CdCl 2 . ( a ) Cells were incubated with 20 μ M CdCl 2 (Cd) for the indicated time. The untreated control is labeled 0 h. Cell lysates were subjected to western blotting using antibodies against E-cadherin, Snail, and actin. Data are from the same experiment shown in . ( b ) Cells transfected with control siRNA or Notch1 siRNA-1 were incubated with or without 20 μ M CdCl 2 (Cd) for 12 h. Cell lysates were subjected to western blotting using antibodies against E-cadherin, Snail, Notch1-NICD, and actin. Data are from the same experiment shown in . ( c ) Cells were incubated with 0.1% DMSO or 25 μ M LY294002 for 1 h and then incubated with or without 20 μ M CdCl 2 (Cd) for 12 h. Cell lysates were subjected to western blotting using antibodies against E-cadherin, Snail, phospho-Akt, total Akt, and actin. ( d ) Cells were incubated with 0.2% DMSO, 20 μ M SB216763 (SB), 25 μ M LY294002 (LY), or 20 μ M SB216763 and 25 μ M LY294002 (SB+LY) for 1 h and then incubated with or without 20 μ M CdCl 2 (Cd) for 12 h. Cell lysates were subjected to western blotting using antibodies against Snail, Notch1-NICD, phospho-Akt, total Akt, and actin. Immunoblots shown are representative of at least three independent experiments

Article Snippet: Antibodies against phospho-EGFR (Tyr1068), total EGFR (D38B1) XP, phospho-Akt (Thr308) (C31E5E), total Akt (pan) (C67E7), phospho-p70 S6 kinase (Thr389) (108D2), phospho-GSK-3 α / β (Ser21/9) (37F11), total GSK3- α / β (D75D3) XP, cleaved Notch1 (Val1744) (D3B8), Notch1 (D1E11) XP, Jagged1 (28H8), Jagged2 (C23D2), Snail (C15D3), E-cadherin (24E10), phospho-p53 (Ser15), and MEK1/2 were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). p53 (DO-1), Notch1 (C-20), actin (I-19), and lamin A/C (14/LaminAC) antibodies were obtained from Santa Cruz Biotechnology.

Techniques: Protein-Protein interactions, Expressing, Incubation, Control, Labeling, Western Blot, Transfection

Journal: Cell Death & Disease

Article Title: Detrimental effects of Notch1 signaling activated by cadmium in renal proximal tubular epithelial cells

doi: 10.1038/cddis.2014.339

Figure Lengend Snippet: Modulation of Notch1 signaling by p53 in HK-2 cells exposed to CdCl 2 . ( a and b ) Cells were incubated with 20 μ M CdCl 2 (Cd) for the indicated time. The untreated control is labeled 0 h. Cell lysates were subjected to western blotting using antibodies against p53, phopho-p53, and actin ( a ). Equal amounts of protein (20 μ g) in nuclear and cytoplasmic extracts were subjected to western blotting using antibodies against p53, MEK1/2, lamin A/C, and actin. MEK1 and lamin A/C served as a loading control for cytoplasmic and nuclear extracts, respectively. Data are from the same experiment shown in ( b ). ( c – e ) Cells were incubated with 0.1% DMSO or 40 μ M pifithrin- α for 1 h and then incubated with or without 20 μ M CdCl 2 (Cd) for 24 h ( c ), 30 h ( d ), or 12 h ( e ). Phase-contrast micrographs were taken ( c ). The viability of cells was determined by trypan blue exclusion assay. Each value is the percentage of trypan blue-positive cells and reflects the mean±S.D. of three experiments with duplicate assays in each experiment. ** P <0.01 versus CdCl 2 -treated cells incubated with DMSO ( d ). Cell lysates were subjected to western blotting using antibodies against Notch1-NICD, Notch1-NTM, and actin ( e ). ( f ) Cells transfected with control siRNA or Notch1 siRNA-1 were incubated with or without 20 μ M CdCl 2 (Cd) for 12 h. Cell lysates were subjected to western blotting using antibodies against p53, phospho-p53, Notch1-NICD, and actin. Immunoblots shown are representative of at least three independent experiments

Article Snippet: Antibodies against phospho-EGFR (Tyr1068), total EGFR (D38B1) XP, phospho-Akt (Thr308) (C31E5E), total Akt (pan) (C67E7), phospho-p70 S6 kinase (Thr389) (108D2), phospho-GSK-3 α / β (Ser21/9) (37F11), total GSK3- α / β (D75D3) XP, cleaved Notch1 (Val1744) (D3B8), Notch1 (D1E11) XP, Jagged1 (28H8), Jagged2 (C23D2), Snail (C15D3), E-cadherin (24E10), phospho-p53 (Ser15), and MEK1/2 were obtained from Cell Signaling Technology, Inc. (Beverly, MA, USA). p53 (DO-1), Notch1 (C-20), actin (I-19), and lamin A/C (14/LaminAC) antibodies were obtained from Santa Cruz Biotechnology.

Techniques: Incubation, Control, Labeling, Western Blot, Trypan Blue Exclusion Assay, Transfection