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Addgene inc human notch1 intracellular domain plasmid

NO sustains CSC stemness through the activation of <t>Notch1.</t> (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 <t>NICD</t> 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).

Human Notch1 Intracellular Domain Plasmid, supplied by Addgene inc, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/human+notch1+intracellular+domain/pmc11550923-36-0-8?v=Addgene+inc
Average 93 stars, based on 4 article reviews

human notch1 intracellular domain plasmid - by Bioz Stars, 2026-07

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1) Product Images from "Nitric oxide facilitates the S‐nitrosylation and deubiquitination of Notch1 protein to maintain cancer stem cells in human NSCLC"

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

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.70203

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).

Figure Legend 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).

Techniques Used: Activation Assay, Western Blot, Expressing

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).

Figure Legend 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).

Techniques Used: Ubiquitin Proteomics, Expressing, Western Blot, Immunoprecipitation

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).

Figure Legend 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).

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

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).

Figure Legend 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).

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

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).

Figure Legend 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).

Techniques Used: Expressing, Immunostaining, Staining, Transfection

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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

Non-NE CDX cells ex vivo are VM-competent and require NOTCH signaling. ( A ) Representative immunoblots of CDX NE and non-NE cell lysates. There are two to three independent replicate tumors per CDX. Tubulin loading control was run subsequently for each marker illustrated on the same blot. ( B ) Representative brightfield images of tubule-forming assay with CDX NE and non-NE cells. There were two to three independent replicate tumors per CDX. Scale bars, 500 μ m. ( C ) Representative immunofluorescence of CDX non-NE cells in tubule-forming assay stained for human mitochondria (yellow) and nuclear DAPI (blue) in ( B ). There were two to three independent replicate tumors per CDX. Scale bars, 100 μ m. ( D-F ) Representative images of HUVECs ( D ), CDX17 ( E ), and CDX30P ( F ) non-NE cells labeled with Cell Tracker Green forming hollow tubules when grown on Matrigel for 72 hours. Confocal microscopy images are illustrated after Z-stack software reconstruction (Imaris). Tubule length and diameter dimensions are illustrated, scale bars 50 μ m. ( G ) Representative images of empty-vector control and NICD expressing CDX31P cells three weeks after dox induction. Scale bars, 250 μ m. ( H ) Percentage of adherent cells in control versus NICD-expressing CDX31P cells three weeks after dox induction in the suspension cells (four replicates from one tumor sample). Data are mean (± SEM) (***p < 0.001 two-tailed unpaired Student’s t test. ( I ) Representative immunoblots in control and NICD CDX31P cells with or without dox. Tubulin loading control was run subsequently for each marker illustrated on the same blot. ( J ) Reverse transcription quantitative PCR analysis of NE ( ASCL1, SYP, NCAm, cHgA, MYCL ) and non-NE ( HEY1, REST, YAP1, FOXC2, MYC ) markers in control versus NICD-expressing CDX31P cells. Mean values are illustrated (black lines) in which each circle represents one independent analysis, error bars are (± SEM). ( K ) Representative images of tubule-forming assay with control and NICD CDX31P cells 3 weeks after dox induction. Scale bars, 200 μ m. There are three independent replicate tumors. CDX, circulating tumor cell-derived explant; DAPI, 4’,6-diamidino-2-phenylindole; dox, doxycycline; HUVECs, human umbilical vein endothelial cells; NE, neuroendocrine; NICD, NOTCH 1 intracellular domain; PCR, polymerase chain reaction; SEM, standard error of the mean; VM, vasculogenic mimicry.

Journal: Journal of thoracic oncology : official publication of the International Association for the Study of Lung Cancer

Article Title: Lineage Plasticity in SCLC Generates Non-Neuroendocrine Cells Primed for Vasculogenic Mimicry

doi: 10.1016/j.jtho.2023.07.012

Figure Lengend Snippet: Non-NE CDX cells ex vivo are VM-competent and require NOTCH signaling. ( A ) Representative immunoblots of CDX NE and non-NE cell lysates. There are two to three independent replicate tumors per CDX. Tubulin loading control was run subsequently for each marker illustrated on the same blot. ( B ) Representative brightfield images of tubule-forming assay with CDX NE and non-NE cells. There were two to three independent replicate tumors per CDX. Scale bars, 500 μ m. ( C ) Representative immunofluorescence of CDX non-NE cells in tubule-forming assay stained for human mitochondria (yellow) and nuclear DAPI (blue) in ( B ). There were two to three independent replicate tumors per CDX. Scale bars, 100 μ m. ( D-F ) Representative images of HUVECs ( D ), CDX17 ( E ), and CDX30P ( F ) non-NE cells labeled with Cell Tracker Green forming hollow tubules when grown on Matrigel for 72 hours. Confocal microscopy images are illustrated after Z-stack software reconstruction (Imaris). Tubule length and diameter dimensions are illustrated, scale bars 50 μ m. ( G ) Representative images of empty-vector control and NICD expressing CDX31P cells three weeks after dox induction. Scale bars, 250 μ m. ( H ) Percentage of adherent cells in control versus NICD-expressing CDX31P cells three weeks after dox induction in the suspension cells (four replicates from one tumor sample). Data are mean (± SEM) (***p < 0.001 two-tailed unpaired Student’s t test. ( I ) Representative immunoblots in control and NICD CDX31P cells with or without dox. Tubulin loading control was run subsequently for each marker illustrated on the same blot. ( J ) Reverse transcription quantitative PCR analysis of NE ( ASCL1, SYP, NCAm, cHgA, MYCL ) and non-NE ( HEY1, REST, YAP1, FOXC2, MYC ) markers in control versus NICD-expressing CDX31P cells. Mean values are illustrated (black lines) in which each circle represents one independent analysis, error bars are (± SEM). ( K ) Representative images of tubule-forming assay with control and NICD CDX31P cells 3 weeks after dox induction. Scale bars, 200 μ m. There are three independent replicate tumors. CDX, circulating tumor cell-derived explant; DAPI, 4’,6-diamidino-2-phenylindole; dox, doxycycline; HUVECs, human umbilical vein endothelial cells; NE, neuroendocrine; NICD, NOTCH 1 intracellular domain; PCR, polymerase chain reaction; SEM, standard error of the mean; VM, vasculogenic mimicry.

Article Snippet: The human NOTCH1 intracellular domain (hN1ICD) doxycycline-inducible expression plasmid (pLIX-hN1ICD) was a gift from Julien Sage (Addgene #91897 [Watertown, MA]).

Techniques: Ex Vivo, Western Blot, Control, Marker, Immunofluorescence, Staining, Labeling, Confocal Microscopy, Software, Plasmid Preparation, Expressing, Suspension, Two Tailed Test, Reverse Transcription, Real-time Polymerase Chain Reaction, Derivative Assay, Polymerase Chain Reaction

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