Journal: Cancers

Article Title: From Deworming to Cancer Therapy: Benzimidazoles in Hematological Malignancies

doi: 10.3390/cancers16203454

Figure Lengend Snippet: Mechanisms of action of anthelmintic benzimidazole-based derivatives as anti-leukemic agents. As a classical anthelmintic, BMDZs inhibit tubulin polymerization ( A ) to induce cell death by mitotic cell arrest ( B ). Albendazole (ABZ) known to accelerate chromosomal missegregation and thus induces loss of heterozygosity in mammalian cells ( C ). Some BMDZs are known to induce terminal differentiation of leukemic blast cells to granulocytes and/or monocytes and thus cause cell death ( D ). Treatment with BMDZs promotes proteasomal degradation of transcriptional factors such as GLI and c-Myb, which play a vital role in leukemia development and progression ( E ). Also, these BMDZs inhibit several oncogenic signaling pathways (Notch1, PI3K/AKT, NF-κB) to achieve remission during the treatment of hematological malignancies ( F ).

Article Snippet: It was also found out that MBZ inhibited the Notch1 intracellular domain (NICD) in CEM/C1 cells.

Techniques: Protein-Protein interactions

Journal: Cell Communication and Signaling : CCS

Article Title: NRN1 interacts with Notch to increase oncogenic STAT3 signaling in melanoma

doi: 10.1186/s12964-024-01632-8

Figure Lengend Snippet: List of plasmids used for transduction or transfection of cells. Company of origin, producer and reference publications included along with description of vector content

Article Snippet: Notch1 intracellular domain-pcw107-V5 was a gift from David Sabatini & Kris Wood (Addgene plasmid # 64622; http://n2t.net/addgene:64622 ; RRID:Addgene_64622) [ ].

Techniques: Transduction, Transfection, Plasmid Preparation, Over Expression, Construct, Luciferase, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nitric oxide facilitates the S‐nitrosylation and deubiquitination of Notch1 protein to maintain cancer stem cells in human NSCLC

doi: 10.1111/jcmm.70203

Figure Lengend Snippet: NO sustains CSC stemness through the activation of Notch1. (A) Immunoblot analysis of Notch1 and Hes1 protein in non‐CSCs and CSCs. (B) The protein expression of Notch1 was analysed in non‐CSCs treated with DETA NONOate (20 μM) for 24 h. (C) CSCs were treated with L‐NAME for 24 h and detected for Notch1 protein with immunoblots. (D, E) CSCs were treated with L‐NAME (100 μM)/1400 W (100 μM) in the presence or absence of the DETA NONOate (20 μM)/GSNO (200 μM) for 24 h. Protein expression of Notch1 and Hes1 were detected by immunoblot. (F–H) CSCs were exposed to L‐NAME (100 μM) with or without the reintroduction of NICD and were analysed for expression of Notch1 protein, stem‐related transcripts and spheres formation. Mean ± SEM from 5 to 9 independent experiments. * p < 0.05, ** p < 0.01 and *** p < 0.001 with paired t ‐test (B, C), unpaired t ‐test (A) and ANOVA plus Turkey's method (D, G, H).

Article Snippet: Human Notch1 intracellular domain plasmid (#130934) was from Addgene.

Techniques: Activation Assay, Western Blot, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nitric oxide facilitates the S‐nitrosylation and deubiquitination of Notch1 protein to maintain cancer stem cells in human NSCLC

doi: 10.1111/jcmm.70203

Figure Lengend Snippet: NO inhibits the ubiquitination of Notch1. (A) mRNA level of Notch1 was measured in CSCs with or without L‐NAME (100 μM) treatment for 24 h. Mean ± SEM from four independent experiments. (B) Notch1 cleavage‐related mRNA expressions in non‐CSCs and CSCs was determined with qPCR. Mean ± SEM from six independent experiments. (C) CSCs were treated with or without L‐NAME (100 μM) for 24 h and detected for Notch1 cleavage‐related mRNA expressions by qPCR. Mean ± SEM from 6 to 8 independent experiments. (D) CSCs were treated with L‐NAME (100 μM) in the presence or absence of the MG132 (10 μM) for 4 h. Protein expression of Notch1 was detected by immunoblot. Mean ± SEM from four independent experiments. (E) Notch1 ubiquitination in non‐CSCs and CSCs was analysed using endogenous immunoprecipitated Notch1 protein. * p < 0.05, ** p < 0.01 with paired t ‐test (A, C), unpaired t ‐test (B) and ANOVA plus Turkey's method (D).

Article Snippet: Human Notch1 intracellular domain plasmid (#130934) was from Addgene.

Techniques: Ubiquitin Proteomics, Expressing, Western Blot, Immunoprecipitation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nitric oxide facilitates the S‐nitrosylation and deubiquitination of Notch1 protein to maintain cancer stem cells in human NSCLC

doi: 10.1111/jcmm.70203

Figure Lengend Snippet: NO inhibits the ubiquitination of Notch1 by facilitating its interaction with UCHL1. (A) Potential DUBs and E3 ligase of Notch1 obtained from the UbiBrowser database. (B, C) DUBs and E3 ligase mRNA expressions in non‐CSCs and CSCs were determined with qPCR. Mean ± SEM from six independent experiments. (D) Immunoblot analysis of UCHL1 protein in non‐CSCs and CSCs. Mean ± SEM from five independent experiments. (E) Co‐immunoprecipitation analysis of Notch1‐UCHL1 interaction in non‐CSCs and CSCs. (F) Genetic knockdown efficiency of UCHL1 in CSCs by lentiviral shRNA transfections. Mean ± SEM from four independent experiments. (G) Immunoblot analysis of Notch1 protein in CSCs transfected with or without the independent UCHL1 shRNAs. Mean ± SEM from four independent experiments. (H) Genetic knockdown efficiency of WWP1, MDM2, USP9X and PSMD7 in CSCs by lentiviral shRNA transfections. Mean ± SEM from 4 to 6 independent experiments. (I) Immunoblot analysis of Notch1 protein in CSCs transfected with or without the independent WWP1, MDM2, USP9X and PSMD7 shRNAs. (J) Immunoprecipitation analysis of ubiquitination of Notch1 in CSCs that were transfected with shUCHL1. (K) CSCs were transfected with UCHL1 shRNAs in the presence or absence of the DETA NONOate (20 μM). The protein of Notch1 was analysed with immunoblot. Mean ± SEM from four independent experiments. (L) Immunoprecipitation analysis of Notch1 ubiquitination in CSCs that were transfected with UCHL1 shRNAs together with DETA NONOate (20 μM). (M) CSCs transfected with UCHL1 shRNAs were analysed for expression of stem‐related transcripts. Mean ± SEM from 5 to 6 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001 with paired t ‐test (M) and ANOVA plus Turkey's method (F–H, K).

Article Snippet: Human Notch1 intracellular domain plasmid (#130934) was from Addgene.

Techniques: Ubiquitin Proteomics, Western Blot, Immunoprecipitation, Knockdown, shRNA, Transfection, Expressing

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nitric oxide facilitates the S‐nitrosylation and deubiquitination of Notch1 protein to maintain cancer stem cells in human NSCLC

doi: 10.1111/jcmm.70203

Figure Lengend Snippet: NO‐facilitated s‐nitrosylation of Notch1 promotes its binding to UCHL1. (A) Biotin switch assay was employed to detect the SNO‐Notch1 levels in both non‐CSCs and CSCs. (B) The SNO‐Notch1 level in DETA NONOate‐treated (20 μM, 24 h) CSCs were detected by biotin switch assay. (C) The SNO‐Notch1 level in L‐NAME‐treated (100 μM, 24 h) CSCs were detected by biotin switch assay. (D) Notch1 protein levels in CSCs with or without ODQ (10 μM, 24 h) were analysed by immunoblotting. Mean ± SEM from four independent experiments. (E) Immunoprecipitation analysis of Notch1 ubiquitination in CSCs in the presence or absence of L‐NAME (100 μM) for 24 h. (F) Immunoprecipitation analysis of Notch1 ubiquitination in CSCs treated with DETA NONOate (20 μM) for 24 h. (G) Co‐immunoprecipitation analysis of Notch1 and UCHL1 interaction in CSCs in the presence or absence of DETA NONOate (20 μM). (H) Co‐immunoprecipitation analysis of Notch1 and UCHL1 interaction in CSCs with or without L‐NAME (100 μM).

Article Snippet: Human Notch1 intracellular domain plasmid (#130934) was from Addgene.

Techniques: Binding Assay, Biotin Switch Assay, Western Blot, Immunoprecipitation, Ubiquitin Proteomics

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nitric oxide facilitates the S‐nitrosylation and deubiquitination of Notch1 protein to maintain cancer stem cells in human NSCLC

doi: 10.1111/jcmm.70203

Figure Lengend Snippet: Targeting UCHL1 and NO downregulates the expression levels of Notch1 and CD133. (A) Representative of PDO‐primary tumour pair with immunostaining for CD31. Nuclei were stained with Hoechst. Scale bar, 20 μm. (B) Representative of PDO‐primary tumour pair with immunostaining for PanCK. Nuclei were stained with Hoechst. Scale bar, 20 μm. (C) Representative HE staining of PDO‐primary tumour pairs. Scale bars, 100 μm. (D, E) PDOs treated with or without DETA NONOate (20 μM) were analysed for CD133 and Notch1 protein. Representatives from six independent experiments. (F, G) PDOs were transfected with UCHL1 shRNAs in the presence or absence of the DETA NONOate (20 μM), followed by analysis of CD133 and Notch1 protein. Representatives from 4 to 5 independent experiments. (H, I) PDOs exposed to 10 Gy x‐rays were treated with L‐NAME (100 μM), followed by analysis of CD133 and Notch1 protein. Representatives from 3 to 5 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 with paired t ‐test (D, E) and ANOVA plus Turkey's method (F–I).

Article Snippet: Human Notch1 intracellular domain plasmid (#130934) was from Addgene.

Techniques: Expressing, Immunostaining, Staining, Transfection

Journal: CytoJournal

Article Title: Roxadustat: A catalyst for diabetic wound healing through re-epithelialization and angiogenesis

doi: 10.25259/Cytojournal_235_2024

Figure Lengend Snippet: Roxadustat (FG-4592) promotes dedifferentiation of keratinocytes and angiogenesis in diabetic mice. (a) Expression levels of integrin β1, K14, K10, K1, and Notch1 NICD evaluated by Western blot in the middle and at the end of wound healing. (b-f) Corresponding quantitative analysis. (g) CD31 and VEGF expression levels evaluated by Western blot. (h and i) Corresponding quantitative analysis. n = 3 in each group. ✶ P < 0.05. NICD: Notch Intracellular Domain, VEGF: Vascular endothelial growth factor, K14: Keratin 14, K10: Keratin 10, K1: Keratin 1.

Article Snippet: The primary antibodies for HIF-1α (1:100, 66730-1-Ig, Proteintech, China) and Notch1 ICD (1:100, PAB35376 , Bioswamp, China) were incubated overnight at 4°C.

Techniques: Expressing, Western Blot

Journal: CytoJournal

Article Title: Roxadustat: A catalyst for diabetic wound healing through re-epithelialization and angiogenesis

doi: 10.25259/Cytojournal_235_2024

Figure Lengend Snippet: Roxadustat (FG-4592) promotes dedifferentiation through interaction between HIF-1α and NICD. (a) Immunofluorescence showing the expression and co-localization of HIF-1α and NICD in HaCaT cells under different conditions; bar = 100 μm. (b) Western blot showing the expression levels of HIF-1α, NICD, K14, and integrin-β1 in HaCaT cells under different conditions. (c-f) Quantitative analysis of WB. n = 3 in each group; ✶ P < 0.05. (g) Co-IP confirming the interaction between HIF-1α and NICD. HIF-1: Hypoxia-inducible factor 1, NICD: Notch intracellular domain, WB: Western blot, Co-IP: Co-immunoprecipitation, K14: Keratin 14.

Article Snippet: The primary antibodies for HIF-1α (1:100, 66730-1-Ig, Proteintech, China) and Notch1 ICD (1:100, PAB35376 , Bioswamp, China) were incubated overnight at 4°C.

Techniques: Immunofluorescence, Expressing, Western Blot, Co-Immunoprecipitation Assay, Immunoprecipitation

Journal: CytoJournal

Article Title: Roxadustat: A catalyst for diabetic wound healing through re-epithelialization and angiogenesis

doi: 10.25259/Cytojournal_235_2024

Figure Lengend Snippet: Schematic model of roxadustat reversing the “high differentiated and low proliferation” state of keratinocytes in diabetic wounds. In normal skin, keratinocytes maintain a relatively stable rhythm of proliferation and differentiation. Injury can activate the process of wound repair, upregulate HIF-1 signal, downregulate Notch1 signal, and make keratinocytes in a state of “high proliferation and low differentiation.” In the context of diabetes, the skin is usually thin and wound healing is delayed, HIF-1 signaling is inhibited, and Notch1 signaling is continuously activated, making keratinocytes in a state of “low proliferation and high differentiation.” FG-4592 upregulates the inhibited HIF-1 signaling and downregulates the hyperactivated Notch1 signaling, which both benefit diabetic wound re-epithelialization. By Fig draw (version 2.0, www.figdraw.com ). HIF-1: Hypoxia-inducible factor 1.

Article Snippet: The primary antibodies for HIF-1α (1:100, 66730-1-Ig, Proteintech, China) and Notch1 ICD (1:100, PAB35376 , Bioswamp, China) were incubated overnight at 4°C.

Techniques: