Western Blot:Article Title: Metastatic small cell lung cancer arises from TP53/RB1-deficient and MYC overproduction hESC-derived PNECs
Article Snippet: .. Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (human) Embryonic stem cell – Rockefeller University Embryonic Stem Cell Line 2 (RUES2) WiCell Research Institute, Inc NIH approval number NIH hESC- 09- 0013, Registration #0013 Passages 7–10 Cell line (human) Embryonic stem cell – ES02 (HES2) WiCell Research Institute, Inc NIH registry Passages 3–7 Cell line (mouse) Mouse embryonic fibroblasts (MEF) Global Stem GSC- 6001G – Antibody Anti- RB1 (Clone 4H1) Cell Signaling Cat# 9309 1:500 (WB) Antibody Anti- P53 (Clone DO- 1) Santa Cruz Biotechnology Cat# Sc- 126 1:500 (WB) Antibody Anti- MYC (Clone D84C12) Cell Signaling Cat# 5605 1:500 (WB) Antibody Anti- HA (Clone 6E2) Cell Signaling Cat# 2367 1:500 (WB) Antibody Anti- CGRP (Clone CD8) Sigma Cat# c9487 1:150 (IHC/IF) Antibody Anti- ASCL1 (Clone 2D9) Sigma Cat# SAB1403577 1:150 (IHC/IF) Antibody Anti- NCAM1/CD56 R&D Systems Cat# AF2408 1:150 (IHC/IF) Antibody Anti- Ki67 (Clone D2H10) Cell Signaling Cat# 9027 1:150 (IHC/IF) Antibody Anti- NEUROD1 (Clone 3H8) Sigma- Aldrich Cat# WH004760M1 1:150 (IHC/IF) Recombinant DNA reagent pSLIK sh human Rb 1534 hyg (Julien Sage) Addgene RRID:Addgene_31500 TET- inducible RB1 shRNA Recombinant DNA reagent FUW- tetO- hMYC (Rudolf Jaenisch) Addgene RRID:Addgene_20723 TET- inducible wild- type MYC Recombinant DNA reagent pLV- tetO- myc T58A (Konrad Hochedlinger) Addgene RRID:Addgene_19763 T58A mutant MYC with 3X- HA tag Chemical compound, drug Paraformaldehyde Sigma P6148 Fixative Chemical compound, drug Triton X- 100 Sigma X- 100 Permeabilization agent Chemical compound, drug Bovine serum albumin (BSA) Life Technologies A9647 – Chemical compound, drug Accutase STEMCELL Technology 07920 – Chemical compound, drug Y- 27632 R&D Systems 1254 – Chemical compound, drug Human BMP4 R&D Systems 314- BP- 010 – Chen, Gardner et al. eLife 2024;13:RP93170. .. DOI: https://doi.org/10.7554/eLife.93170 12 of 20 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Chemical compound, drug Human bFGF R&D Systems 233- FB – Chemical compound, drug Human Activin A R&D Systems 338- AC – Chemical compound, drug Dorsomorphin dihydrochloride Sigma AMBH2D6FB2F5 – Chemical compound, drug SB431542 R&D Systems TB1614- GMP – Chemical compound, drug IWP2 Sigma I0536 – Chemical compound, drug CHIR99021 R&D Systems 5439- DK – Chemical compound, drug Human FGF10 R&D Systems Qk003- 0050 – Chemical compound, drug Human FGF7 R&D Systems 251- KG – Chemical compound, drug All- trans retinoic acid (ATRA) Sigma 554720 – Chemical compound, drug Dexamethasone Sigma 265005 – Chemical compound, drug 8- Bromo- cAMP Sigma- Aldrich TA9H9A9A7499 – Chemical compound, drug 3,7- Dihydro- 1- methyl- 3- (2- methylpropyl)- 1H- purine- 2,6- dione (IBMX) Sigma- Aldrich TA9H98DB18B1 – Chemical compound, drug DAPT Sigma- Aldrich D5942 – Chemical compound, drug Monothioglycerol Sigma M6145 Other mTeSR1 STEMCELL Technologies 85850 hESC maintenance for transduced hESC culture Other Matrigel Corning 354277 ECM- coated plates for maturation Other DMEM/F- 12 Gibco 10565- 018 Medium Other N2 Gibco 17502001 Supplement Other B27 Gibco 17504044 Supplement Other Ascorbic acid Sigma 1043003 Other GlutaMAX Life Technologies 35050061 Supplement Other 0.05% Trypsin/0.02% EDTA Life Technologies 25300062 Enzyme Continued Generation of lung cells, including PNECs, from hESCs Protocols for maintenance of hESCs and generation of PNECs and other lung cells were modified from previous studies (Huang et al., 2015; Huang et al., 2014; Chen et al., 2019).
Immunohistochemistry:Article Title: Metastatic small cell lung cancer arises from TP53/RB1-deficient and MYC overproduction hESC-derived PNECs
Article Snippet: .. Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (human) Embryonic stem cell – Rockefeller University Embryonic Stem Cell Line 2 (RUES2) WiCell Research Institute, Inc NIH approval number NIH hESC- 09- 0013, Registration #0013 Passages 7–10 Cell line (human) Embryonic stem cell – ES02 (HES2) WiCell Research Institute, Inc NIH registry Passages 3–7 Cell line (mouse) Mouse embryonic fibroblasts (MEF) Global Stem GSC- 6001G – Antibody Anti- RB1 (Clone 4H1) Cell Signaling Cat# 9309 1:500 (WB) Antibody Anti- P53 (Clone DO- 1) Santa Cruz Biotechnology Cat# Sc- 126 1:500 (WB) Antibody Anti- MYC (Clone D84C12) Cell Signaling Cat# 5605 1:500 (WB) Antibody Anti- HA (Clone 6E2) Cell Signaling Cat# 2367 1:500 (WB) Antibody Anti- CGRP (Clone CD8) Sigma Cat# c9487 1:150 (IHC/IF) Antibody Anti- ASCL1 (Clone 2D9) Sigma Cat# SAB1403577 1:150 (IHC/IF) Antibody Anti- NCAM1/CD56 R&D Systems Cat# AF2408 1:150 (IHC/IF) Antibody Anti- Ki67 (Clone D2H10) Cell Signaling Cat# 9027 1:150 (IHC/IF) Antibody Anti- NEUROD1 (Clone 3H8) Sigma- Aldrich Cat# WH004760M1 1:150 (IHC/IF) Recombinant DNA reagent pSLIK sh human Rb 1534 hyg (Julien Sage) Addgene RRID:Addgene_31500 TET- inducible RB1 shRNA Recombinant DNA reagent FUW- tetO- hMYC (Rudolf Jaenisch) Addgene RRID:Addgene_20723 TET- inducible wild- type MYC Recombinant DNA reagent pLV- tetO- myc T58A (Konrad Hochedlinger) Addgene RRID:Addgene_19763 T58A mutant MYC with 3X- HA tag Chemical compound, drug Paraformaldehyde Sigma P6148 Fixative Chemical compound, drug Triton X- 100 Sigma X- 100 Permeabilization agent Chemical compound, drug Bovine serum albumin (BSA) Life Technologies A9647 – Chemical compound, drug Accutase STEMCELL Technology 07920 – Chemical compound, drug Y- 27632 R&D Systems 1254 – Chemical compound, drug Human BMP4 R&D Systems 314- BP- 010 – Chen, Gardner et al. eLife 2024;13:RP93170. .. DOI: https://doi.org/10.7554/eLife.93170 12 of 20 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Chemical compound, drug Human bFGF R&D Systems 233- FB – Chemical compound, drug Human Activin A R&D Systems 338- AC – Chemical compound, drug Dorsomorphin dihydrochloride Sigma AMBH2D6FB2F5 – Chemical compound, drug SB431542 R&D Systems TB1614- GMP – Chemical compound, drug IWP2 Sigma I0536 – Chemical compound, drug CHIR99021 R&D Systems 5439- DK – Chemical compound, drug Human FGF10 R&D Systems Qk003- 0050 – Chemical compound, drug Human FGF7 R&D Systems 251- KG – Chemical compound, drug All- trans retinoic acid (ATRA) Sigma 554720 – Chemical compound, drug Dexamethasone Sigma 265005 – Chemical compound, drug 8- Bromo- cAMP Sigma- Aldrich TA9H9A9A7499 – Chemical compound, drug 3,7- Dihydro- 1- methyl- 3- (2- methylpropyl)- 1H- purine- 2,6- dione (IBMX) Sigma- Aldrich TA9H98DB18B1 – Chemical compound, drug DAPT Sigma- Aldrich D5942 – Chemical compound, drug Monothioglycerol Sigma M6145 Other mTeSR1 STEMCELL Technologies 85850 hESC maintenance for transduced hESC culture Other Matrigel Corning 354277 ECM- coated plates for maturation Other DMEM/F- 12 Gibco 10565- 018 Medium Other N2 Gibco 17502001 Supplement Other B27 Gibco 17504044 Supplement Other Ascorbic acid Sigma 1043003 Other GlutaMAX Life Technologies 35050061 Supplement Other 0.05% Trypsin/0.02% EDTA Life Technologies 25300062 Enzyme Continued Generation of lung cells, including PNECs, from hESCs Protocols for maintenance of hESCs and generation of PNECs and other lung cells were modified from previous studies (Huang et al., 2015; Huang et al., 2014; Chen et al., 2019).
Recombinant:Article Title: Metastatic small cell lung cancer arises from TP53/RB1-deficient and MYC overproduction hESC-derived PNECs
Article Snippet: .. Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (human) Embryonic stem cell – Rockefeller University Embryonic Stem Cell Line 2 (RUES2) WiCell Research Institute, Inc NIH approval number NIH hESC- 09- 0013, Registration #0013 Passages 7–10 Cell line (human) Embryonic stem cell – ES02 (HES2) WiCell Research Institute, Inc NIH registry Passages 3–7 Cell line (mouse) Mouse embryonic fibroblasts (MEF) Global Stem GSC- 6001G – Antibody Anti- RB1 (Clone 4H1) Cell Signaling Cat# 9309 1:500 (WB) Antibody Anti- P53 (Clone DO- 1) Santa Cruz Biotechnology Cat# Sc- 126 1:500 (WB) Antibody Anti- MYC (Clone D84C12) Cell Signaling Cat# 5605 1:500 (WB) Antibody Anti- HA (Clone 6E2) Cell Signaling Cat# 2367 1:500 (WB) Antibody Anti- CGRP (Clone CD8) Sigma Cat# c9487 1:150 (IHC/IF) Antibody Anti- ASCL1 (Clone 2D9) Sigma Cat# SAB1403577 1:150 (IHC/IF) Antibody Anti- NCAM1/CD56 R&D Systems Cat# AF2408 1:150 (IHC/IF) Antibody Anti- Ki67 (Clone D2H10) Cell Signaling Cat# 9027 1:150 (IHC/IF) Antibody Anti- NEUROD1 (Clone 3H8) Sigma- Aldrich Cat# WH004760M1 1:150 (IHC/IF) Recombinant DNA reagent pSLIK sh human Rb 1534 hyg (Julien Sage) Addgene RRID:Addgene_31500 TET- inducible RB1 shRNA Recombinant DNA reagent FUW- tetO- hMYC (Rudolf Jaenisch) Addgene RRID:Addgene_20723 TET- inducible wild- type MYC Recombinant DNA reagent pLV- tetO- myc T58A (Konrad Hochedlinger) Addgene RRID:Addgene_19763 T58A mutant MYC with 3X- HA tag Chemical compound, drug Paraformaldehyde Sigma P6148 Fixative Chemical compound, drug Triton X- 100 Sigma X- 100 Permeabilization agent Chemical compound, drug Bovine serum albumin (BSA) Life Technologies A9647 – Chemical compound, drug Accutase STEMCELL Technology 07920 – Chemical compound, drug Y- 27632 R&D Systems 1254 – Chemical compound, drug Human BMP4 R&D Systems 314- BP- 010 – Chen, Gardner et al. eLife 2024;13:RP93170. .. DOI: https://doi.org/10.7554/eLife.93170 12 of 20 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Chemical compound, drug Human bFGF R&D Systems 233- FB – Chemical compound, drug Human Activin A R&D Systems 338- AC – Chemical compound, drug Dorsomorphin dihydrochloride Sigma AMBH2D6FB2F5 – Chemical compound, drug SB431542 R&D Systems TB1614- GMP – Chemical compound, drug IWP2 Sigma I0536 – Chemical compound, drug CHIR99021 R&D Systems 5439- DK – Chemical compound, drug Human FGF10 R&D Systems Qk003- 0050 – Chemical compound, drug Human FGF7 R&D Systems 251- KG – Chemical compound, drug All- trans retinoic acid (ATRA) Sigma 554720 – Chemical compound, drug Dexamethasone Sigma 265005 – Chemical compound, drug 8- Bromo- cAMP Sigma- Aldrich TA9H9A9A7499 – Chemical compound, drug 3,7- Dihydro- 1- methyl- 3- (2- methylpropyl)- 1H- purine- 2,6- dione (IBMX) Sigma- Aldrich TA9H98DB18B1 – Chemical compound, drug DAPT Sigma- Aldrich D5942 – Chemical compound, drug Monothioglycerol Sigma M6145 Other mTeSR1 STEMCELL Technologies 85850 hESC maintenance for transduced hESC culture Other Matrigel Corning 354277 ECM- coated plates for maturation Other DMEM/F- 12 Gibco 10565- 018 Medium Other N2 Gibco 17502001 Supplement Other B27 Gibco 17504044 Supplement Other Ascorbic acid Sigma 1043003 Other GlutaMAX Life Technologies 35050061 Supplement Other 0.05% Trypsin/0.02% EDTA Life Technologies 25300062 Enzyme Continued Generation of lung cells, including PNECs, from hESCs Protocols for maintenance of hESCs and generation of PNECs and other lung cells were modified from previous studies (Huang et al., 2015; Huang et al., 2014; Chen et al., 2019).
shRNA:Article Title: Metastatic small cell lung cancer arises from TP53/RB1-deficient and MYC overproduction hESC-derived PNECs
Article Snippet: .. Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (human) Embryonic stem cell – Rockefeller University Embryonic Stem Cell Line 2 (RUES2) WiCell Research Institute, Inc NIH approval number NIH hESC- 09- 0013, Registration #0013 Passages 7–10 Cell line (human) Embryonic stem cell – ES02 (HES2) WiCell Research Institute, Inc NIH registry Passages 3–7 Cell line (mouse) Mouse embryonic fibroblasts (MEF) Global Stem GSC- 6001G – Antibody Anti- RB1 (Clone 4H1) Cell Signaling Cat# 9309 1:500 (WB) Antibody Anti- P53 (Clone DO- 1) Santa Cruz Biotechnology Cat# Sc- 126 1:500 (WB) Antibody Anti- MYC (Clone D84C12) Cell Signaling Cat# 5605 1:500 (WB) Antibody Anti- HA (Clone 6E2) Cell Signaling Cat# 2367 1:500 (WB) Antibody Anti- CGRP (Clone CD8) Sigma Cat# c9487 1:150 (IHC/IF) Antibody Anti- ASCL1 (Clone 2D9) Sigma Cat# SAB1403577 1:150 (IHC/IF) Antibody Anti- NCAM1/CD56 R&D Systems Cat# AF2408 1:150 (IHC/IF) Antibody Anti- Ki67 (Clone D2H10) Cell Signaling Cat# 9027 1:150 (IHC/IF) Antibody Anti- NEUROD1 (Clone 3H8) Sigma- Aldrich Cat# WH004760M1 1:150 (IHC/IF) Recombinant DNA reagent pSLIK sh human Rb 1534 hyg (Julien Sage) Addgene RRID:Addgene_31500 TET- inducible RB1 shRNA Recombinant DNA reagent FUW- tetO- hMYC (Rudolf Jaenisch) Addgene RRID:Addgene_20723 TET- inducible wild- type MYC Recombinant DNA reagent pLV- tetO- myc T58A (Konrad Hochedlinger) Addgene RRID:Addgene_19763 T58A mutant MYC with 3X- HA tag Chemical compound, drug Paraformaldehyde Sigma P6148 Fixative Chemical compound, drug Triton X- 100 Sigma X- 100 Permeabilization agent Chemical compound, drug Bovine serum albumin (BSA) Life Technologies A9647 – Chemical compound, drug Accutase STEMCELL Technology 07920 – Chemical compound, drug Y- 27632 R&D Systems 1254 – Chemical compound, drug Human BMP4 R&D Systems 314- BP- 010 – Chen, Gardner et al. eLife 2024;13:RP93170. .. DOI: https://doi.org/10.7554/eLife.93170 12 of 20 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Chemical compound, drug Human bFGF R&D Systems 233- FB – Chemical compound, drug Human Activin A R&D Systems 338- AC – Chemical compound, drug Dorsomorphin dihydrochloride Sigma AMBH2D6FB2F5 – Chemical compound, drug SB431542 R&D Systems TB1614- GMP – Chemical compound, drug IWP2 Sigma I0536 – Chemical compound, drug CHIR99021 R&D Systems 5439- DK – Chemical compound, drug Human FGF10 R&D Systems Qk003- 0050 – Chemical compound, drug Human FGF7 R&D Systems 251- KG – Chemical compound, drug All- trans retinoic acid (ATRA) Sigma 554720 – Chemical compound, drug Dexamethasone Sigma 265005 – Chemical compound, drug 8- Bromo- cAMP Sigma- Aldrich TA9H9A9A7499 – Chemical compound, drug 3,7- Dihydro- 1- methyl- 3- (2- methylpropyl)- 1H- purine- 2,6- dione (IBMX) Sigma- Aldrich TA9H98DB18B1 – Chemical compound, drug DAPT Sigma- Aldrich D5942 – Chemical compound, drug Monothioglycerol Sigma M6145 Other mTeSR1 STEMCELL Technologies 85850 hESC maintenance for transduced hESC culture Other Matrigel Corning 354277 ECM- coated plates for maturation Other DMEM/F- 12 Gibco 10565- 018 Medium Other N2 Gibco 17502001 Supplement Other B27 Gibco 17504044 Supplement Other Ascorbic acid Sigma 1043003 Other GlutaMAX Life Technologies 35050061 Supplement Other 0.05% Trypsin/0.02% EDTA Life Technologies 25300062 Enzyme Continued Generation of lung cells, including PNECs, from hESCs Protocols for maintenance of hESCs and generation of PNECs and other lung cells were modified from previous studies (Huang et al., 2015; Huang et al., 2014; Chen et al., 2019).
Mutagenesis:Article Title: Metastatic small cell lung cancer arises from TP53/RB1-deficient and MYC overproduction hESC-derived PNECs
Article Snippet: .. Reagent type (species) or resource Designation Source or reference Identifiers Additional information Cell line (human) Embryonic stem cell – Rockefeller University Embryonic Stem Cell Line 2 (RUES2) WiCell Research Institute, Inc NIH approval number NIH hESC- 09- 0013, Registration #0013 Passages 7–10 Cell line (human) Embryonic stem cell – ES02 (HES2) WiCell Research Institute, Inc NIH registry Passages 3–7 Cell line (mouse) Mouse embryonic fibroblasts (MEF) Global Stem GSC- 6001G – Antibody Anti- RB1 (Clone 4H1) Cell Signaling Cat# 9309 1:500 (WB) Antibody Anti- P53 (Clone DO- 1) Santa Cruz Biotechnology Cat# Sc- 126 1:500 (WB) Antibody Anti- MYC (Clone D84C12) Cell Signaling Cat# 5605 1:500 (WB) Antibody Anti- HA (Clone 6E2) Cell Signaling Cat# 2367 1:500 (WB) Antibody Anti- CGRP (Clone CD8) Sigma Cat# c9487 1:150 (IHC/IF) Antibody Anti- ASCL1 (Clone 2D9) Sigma Cat# SAB1403577 1:150 (IHC/IF) Antibody Anti- NCAM1/CD56 R&D Systems Cat# AF2408 1:150 (IHC/IF) Antibody Anti- Ki67 (Clone D2H10) Cell Signaling Cat# 9027 1:150 (IHC/IF) Antibody Anti- NEUROD1 (Clone 3H8) Sigma- Aldrich Cat# WH004760M1 1:150 (IHC/IF) Recombinant DNA reagent pSLIK sh human Rb 1534 hyg (Julien Sage) Addgene RRID:Addgene_31500 TET- inducible RB1 shRNA Recombinant DNA reagent FUW- tetO- hMYC (Rudolf Jaenisch) Addgene RRID:Addgene_20723 TET- inducible wild- type MYC Recombinant DNA reagent pLV- tetO- myc T58A (Konrad Hochedlinger) Addgene RRID:Addgene_19763 T58A mutant MYC with 3X- HA tag Chemical compound, drug Paraformaldehyde Sigma P6148 Fixative Chemical compound, drug Triton X- 100 Sigma X- 100 Permeabilization agent Chemical compound, drug Bovine serum albumin (BSA) Life Technologies A9647 – Chemical compound, drug Accutase STEMCELL Technology 07920 – Chemical compound, drug Y- 27632 R&D Systems 1254 – Chemical compound, drug Human BMP4 R&D Systems 314- BP- 010 – Chen, Gardner et al. eLife 2024;13:RP93170. .. DOI: https://doi.org/10.7554/eLife.93170 12 of 20 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Chemical compound, drug Human bFGF R&D Systems 233- FB – Chemical compound, drug Human Activin A R&D Systems 338- AC – Chemical compound, drug Dorsomorphin dihydrochloride Sigma AMBH2D6FB2F5 – Chemical compound, drug SB431542 R&D Systems TB1614- GMP – Chemical compound, drug IWP2 Sigma I0536 – Chemical compound, drug CHIR99021 R&D Systems 5439- DK – Chemical compound, drug Human FGF10 R&D Systems Qk003- 0050 – Chemical compound, drug Human FGF7 R&D Systems 251- KG – Chemical compound, drug All- trans retinoic acid (ATRA) Sigma 554720 – Chemical compound, drug Dexamethasone Sigma 265005 – Chemical compound, drug 8- Bromo- cAMP Sigma- Aldrich TA9H9A9A7499 – Chemical compound, drug 3,7- Dihydro- 1- methyl- 3- (2- methylpropyl)- 1H- purine- 2,6- dione (IBMX) Sigma- Aldrich TA9H98DB18B1 – Chemical compound, drug DAPT Sigma- Aldrich D5942 – Chemical compound, drug Monothioglycerol Sigma M6145 Other mTeSR1 STEMCELL Technologies 85850 hESC maintenance for transduced hESC culture Other Matrigel Corning 354277 ECM- coated plates for maturation Other DMEM/F- 12 Gibco 10565- 018 Medium Other N2 Gibco 17502001 Supplement Other B27 Gibco 17504044 Supplement Other Ascorbic acid Sigma 1043003 Other GlutaMAX Life Technologies 35050061 Supplement Other 0.05% Trypsin/0.02% EDTA Life Technologies 25300062 Enzyme Continued Generation of lung cells, including PNECs, from hESCs Protocols for maintenance of hESCs and generation of PNECs and other lung cells were modified from previous studies (Huang et al., 2015; Huang et al., 2014; Chen et al., 2019).
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