Derivative Assay:Article Title: Antagonism among DUX family members evolved from an ancestral toxic single homeodomain protein
Article Snippet: Control LHCN-M2 immortalized human myoblasts and FSHD (M008) immortalized human myoblasts were cultured in F10 medium (HyClone) with 20% FBS (Peak Serum), 2-mercaptoethanol 1x (Gibco), 10 −9 M dexamethasone (Sigma), 10 ng/mL bFGF (Peprotech), Glutamax (Gibco), and Penicillin/Streptomycin (P/S, Gibco) at 37°C and 5% CO 2 . .. Immortalized M008 cells were derived by transducing primary myoblasts obtained from CD56 sorting of mononuclear cells from a muscle biopsy of an individual with FSHD under a protocol approved by the University of Minnesota IRB, with pbabe-cyclinD1+CDK4R24C (gift of Christopher Counter, Addgene) and pLV-hTERT-IRES-hygro (gift of Tobias Meyer, Addgene) . ..
Article Title: Antagonism among DUX family members evolved from an ancestral toxic single homeodomain protein
Article Snippet: Control LHCN-M2 immortalized human myoblasts and FSHD (M008) immortalized human myoblasts were cultured in F10 medium (HyClone) with 20% FBS (Peak Serum), 2-mercaptoethanol 1x (Gibco), 10 −9 M dexamethasone (Sigma), 10 ng/mL bFGF (Peprotech), Glutamax (Gibco), and Penicillin/Streptomycin (P/S, Gibco) at 37°C and 5% CO 2 . .. Immortalized M008 cells were derived by transducing primary myoblasts obtained from CD56 sorting of mononuclear cells from a muscle biopsy of an individual with FSHD under a protocol approved by the University of Minnesota IRB, with pbabe-cyclinD1+CDK4R24C (gift of Christopher Counter, Addgene) and pLV-hTERT-IRES-hygro (gift of Tobias Meyer, Addgene) . ..
Article Title: Antagonism among DUX family members evolved from an ancestral toxic single homeodomain protein.
Article Snippet: Control LHCN-M2 immortalized human myoblasts38 and FSHD (M008) immortalized human myoblasts were cultured in F10 medium (HyClone) with 20% FBS (Peak Serum), 2-mercaptoethanol 1x (Gibco), 10-9 M dexamethasone (Sigma), 10 ng/mL bFGF (Peprotech), Glutamax (Gibco), and Penicillin/Streptomycin (P/S, Gibco) at 37 C and 5% CO2. .. Immortalized M008 cells were derived by transducing primary myoblasts obtained from CD56 sorting of mononuclear cells from a muscle biopsy of an individual with FSHD under a protocol approved by the University of Minnesota IRB, with pbabe-cyclinD1+CDK4R24C (gift of Christopher Counter, Addgene)39 and pLV-hTERT-IRES-hygro (gift of TobiasMeyer, Addgene).40 TheM007iDUX4 andM008iDUXA cell lines were generated fromM007 andM008 immortalizedmyoblasts, which were derived by transduction of primary myoblasts from control and FSHD donors respectively, with two lentivectors, FUGW-rtTA and pSAM2-DUXA as previously described.13 The LHCN-M2iDUX4&DUXA cell line was generated by transducing LHCN-M2iDUX4 cells with pSAM-DUXA-ires-GFP lentivirus. .. GFP positive cells were FACS sorted two days post infection using a BD FACSAria (BD Biosciences, Franklin Lakes, NJ).
Article Title: Antagonism among DUX family members evolved from an ancestral toxic single homeodomain protein
Article Snippet: Control LHCN-M2 immortalized human myoblasts and FSHD (M008) immortalized human myoblasts were cultured in F10 medium (HyClone) with 20% FBS (Peak Serum), 2-mercaptoethanol 1x (Gibco), 10 -9 M dexamethasone (Sigma), 10 ng/mL bFGF (Peprotech), Glutamax (Gibco), and Penicillin/Streptomycin (P/S, Gibco) at 37°C and 5% CO 2 . .. Immortalized M008 cells were derived by transducing primary myoblasts obtained from CD56 sorting of mononuclear cells from a muscle biopsy of an individual with FSHD under a protocol approved by the University of Minnesota IRB, with pbabe-cyclinD1+CDK4R24C (gift of Christopher Counter, Addgene) and pLV-hTERT-IRES-hygro (gift of Tobias Meyer, Addgene). ..
Isolation:Article Title: Hyper-active RAS/MAPK introduces cancer-specific mitotic vulnerabilities
Article Snippet: .. Ref. 14 See Supplementary Table 3 for more details Nucleic Acid Kits NEBNext Ultra II Directional Library Prep Kit for Illumina New England BioLabs E7760S Nucleic Acid Kits NEBNext Poly(A) mRNA Magnetic Isolation Module New England BioLabs E7490S Nucleic Acid Kits RNeasy mini kit Qiagen 74104 Nucleic Acid Kits iScript cDNA synthesis kit Bio-rad 1708890 Plasmid hTert/mp53DD Addgene 11128 See Supplementary Table 2 for more details Plasmid CCND1/Cdk4R24C Addgene 11129 See Supplementary Table 2 for more details Plasmid c-mycT58A/HRasG12V Addgene 11130 See Supplementary Table 2 for more details Plasmid myr-FLAG-Akt Addgene 15294 See Supplementary Table 2 for more details Plasmid MekDD Addgene 15268 See Supplementary Table 2 for more details Plasmid H-RasV12 Addgene 18749 See Supplementary Table 2 for more details Plasmid H2B-EGFP Addgene 21210 See Supplementary Table 2 for more details Plasmid EGFP-BubR1WT This study See Supplementary Table 2 for more details Plasmid EGFP-BubR13A This study See Supplementary Table 2 for more details Plasmid EGFP-Mis12-KARD3D This study See Supplementary Table 2 for more details Plasmid pLEP3_EGFP-BubR1WT This study See Supplementary Table 2 for more details Plasmid pLEP3_EGFP-BubR13A This study See Supplementary Table 2 for more details Primary Antibody anti-pT202/Y204 ERK1/2 [rabbit polyclonal] GeneTex GTX24819 Primary Antibody anti-pS133 CREB (1B6) [mouse monoclonal] Cell Signaling 9196 Primary Antibody anti-histone H3 [rabbit polyclonal] Abcam ab1791 Primary Antibody anti-ERK1/2 [rabbit monoclonal] Cell Signaling 4695 Primary Antibody anti-BubR1 (8G1) [mouse monoclonal] Millipore Sigma MAB3612 1:500 Primary Antibody anti-Hec1 (9G3) [mouse monoclonal] GeneTex GTX70268 2 μg/mL Primary Antibody anti-centromere antibody (ACA) [human polyclonal] Antibodies Inc. 15-235 1:600 Primary Antibody anti-pS55 Hec1 [rabbit purified polyclonal] Supp. .. Ref. 14 See Supplementary Table 3 for more details Nucleic Acid Kits NEBNext Ultra II Directional Library Prep Kit for Illumina New England BioLabs E7760S Nucleic Acid Kits NEBNext Poly(A) mRNA Magnetic Isolation Module New England BioLabs E7490S Nucleic Acid Kits RNeasy mini kit Qiagen 74104 Nucleic Acid Kits iScript cDNA synthesis kit Bio-rad 1708890 Plasmid hTert/mp53DD Addgene 11128 See Supplementary Table 2 for more details Plasmid CCND1/Cdk4R24C Addgene 11129 See Supplementary Table 2 for more details Plasmid c-mycT58A/HRasG12V Addgene 11130 See Supplementary Table 2 for more details Plasmid myr-FLAG-Akt Addgene 15294 See Supplementary Table 2 for more details Plasmid MekDD Addgene 15268 See Supplementary Table 2 for more details Plasmid H-RasV12 Addgene 18749 See Supplementary Table 2 for more details Plasmid H2B-EGFP Addgene 21210 See Supplementary Table 2 for more details Plasmid EGFP-BubR1WT This study See Supplementary Table 2 for more details Plasmid EGFP-BubR13A This study See Supplementary Table 2 for more details Plasmid EGFP-Mis12-KARD3D This study See Supplementary Table 2 for more details Plasmid pLEP3_EGFP-BubR1WT This study See Supplementary Table 2 for more details Plasmid pLEP3_EGFP-BubR13A This study See Supplementary Table 2 for more details Primary Antibody anti-pT202/Y204 ERK1/2 [rabbit polyclonal] GeneTex GTX24819 Primary Antibody anti-pS133 CREB (1B6) [mouse monoclonal] Cell Signaling 9196 Primary Antibody anti-histone H3 [rabbit polyclonal] Abcam ab1791 Primary Antibody anti-ERK1/2 [rabbit monoclonal] Cell Signaling 4695 Primary Antibody anti-BubR1 (8G1) [mouse monoclonal] Millipore Sigma MAB3612 1:500 Primary Antibody anti-Hec1 (9G3) [mouse monoclonal] GeneTex GTX70268 2 μg/mL Primary Antibody anti-centromere antibody (ACA) [human polyclonal] Antibodies Inc. 15-235 1:600 Primary Antibody anti-pS55 Hec1 [rabbit purified polyclonal] Supp.
cDNA Synthesis:Article Title: Hyper-active RAS/MAPK introduces cancer-specific mitotic vulnerabilities
Article Snippet: .. Ref. 14 See Supplementary Table 3 for more details Nucleic Acid Kits NEBNext Ultra II Directional Library Prep Kit for Illumina New England BioLabs E7760S Nucleic Acid Kits NEBNext Poly(A) mRNA Magnetic Isolation Module New England BioLabs E7490S Nucleic Acid Kits RNeasy mini kit Qiagen 74104 Nucleic Acid Kits iScript cDNA synthesis kit Bio-rad 1708890 Plasmid hTert/mp53DD Addgene 11128 See Supplementary Table 2 for more details Plasmid CCND1/Cdk4R24C Addgene 11129 See Supplementary Table 2 for more details Plasmid c-mycT58A/HRasG12V Addgene 11130 See Supplementary Table 2 for more details Plasmid myr-FLAG-Akt Addgene 15294 See Supplementary Table 2 for more details Plasmid MekDD Addgene 15268 See Supplementary Table 2 for more details Plasmid H-RasV12 Addgene 18749 See Supplementary Table 2 for more details Plasmid H2B-EGFP Addgene 21210 See Supplementary Table 2 for more details Plasmid EGFP-BubR1WT This study See Supplementary Table 2 for more details Plasmid EGFP-BubR13A This study See Supplementary Table 2 for more details Plasmid EGFP-Mis12-KARD3D This study See Supplementary Table 2 for more details Plasmid pLEP3_EGFP-BubR1WT This study See Supplementary Table 2 for more details Plasmid pLEP3_EGFP-BubR13A This study See Supplementary Table 2 for more details Primary Antibody anti-pT202/Y204 ERK1/2 [rabbit polyclonal] GeneTex GTX24819 Primary Antibody anti-pS133 CREB (1B6) [mouse monoclonal] Cell Signaling 9196 Primary Antibody anti-histone H3 [rabbit polyclonal] Abcam ab1791 Primary Antibody anti-ERK1/2 [rabbit monoclonal] Cell Signaling 4695 Primary Antibody anti-BubR1 (8G1) [mouse monoclonal] Millipore Sigma MAB3612 1:500 Primary Antibody anti-Hec1 (9G3) [mouse monoclonal] GeneTex GTX70268 2 μg/mL Primary Antibody anti-centromere antibody (ACA) [human polyclonal] Antibodies Inc. 15-235 1:600 Primary Antibody anti-pS55 Hec1 [rabbit purified polyclonal] Supp. .. Ref. 14 See Supplementary Table 3 for more details Nucleic Acid Kits NEBNext Ultra II Directional Library Prep Kit for Illumina New England BioLabs E7760S Nucleic Acid Kits NEBNext Poly(A) mRNA Magnetic Isolation Module New England BioLabs E7490S Nucleic Acid Kits RNeasy mini kit Qiagen 74104 Nucleic Acid Kits iScript cDNA synthesis kit Bio-rad 1708890 Plasmid hTert/mp53DD Addgene 11128 See Supplementary Table 2 for more details Plasmid CCND1/Cdk4R24C Addgene 11129 See Supplementary Table 2 for more details Plasmid c-mycT58A/HRasG12V Addgene 11130 See Supplementary Table 2 for more details Plasmid myr-FLAG-Akt Addgene 15294 See Supplementary Table 2 for more details Plasmid MekDD Addgene 15268 See Supplementary Table 2 for more details Plasmid H-RasV12 Addgene 18749 See Supplementary Table 2 for more details Plasmid H2B-EGFP Addgene 21210 See Supplementary Table 2 for more details Plasmid EGFP-BubR1WT This study See Supplementary Table 2 for more details Plasmid EGFP-BubR13A This study See Supplementary Table 2 for more details Plasmid EGFP-Mis12-KARD3D This study See Supplementary Table 2 for more details Plasmid pLEP3_EGFP-BubR1WT This study See Supplementary Table 2 for more details Plasmid pLEP3_EGFP-BubR13A This study See Supplementary Table 2 for more details Primary Antibody anti-pT202/Y204 ERK1/2 [rabbit polyclonal] GeneTex GTX24819 Primary Antibody anti-pS133 CREB (1B6) [mouse monoclonal] Cell Signaling 9196 Primary Antibody anti-histone H3 [rabbit polyclonal] Abcam ab1791 Primary Antibody anti-ERK1/2 [rabbit monoclonal] Cell Signaling 4695 Primary Antibody anti-BubR1 (8G1) [mouse monoclonal] Millipore Sigma MAB3612 1:500 Primary Antibody anti-Hec1 (9G3) [mouse monoclonal] GeneTex GTX70268 2 μg/mL Primary Antibody anti-centromere antibody (ACA) [human polyclonal] Antibodies Inc. 15-235 1:600 Primary Antibody anti-pS55 Hec1 [rabbit purified polyclonal] Supp.
Plasmid Preparation:Article Title: Hyper-active RAS/MAPK introduces cancer-specific mitotic vulnerabilities
Article Snippet: .. Ref. 14 See Supplementary Table 3 for more details Nucleic Acid Kits NEBNext Ultra II Directional Library Prep Kit for Illumina New England BioLabs E7760S Nucleic Acid Kits NEBNext Poly(A) mRNA Magnetic Isolation Module New England BioLabs E7490S Nucleic Acid Kits RNeasy mini kit Qiagen 74104 Nucleic Acid Kits iScript cDNA synthesis kit Bio-rad 1708890 Plasmid hTert/mp53DD Addgene 11128 See Supplementary Table 2 for more details Plasmid CCND1/Cdk4R24C Addgene 11129 See Supplementary Table 2 for more details Plasmid c-mycT58A/HRasG12V Addgene 11130 See Supplementary Table 2 for more details Plasmid myr-FLAG-Akt Addgene 15294 See Supplementary Table 2 for more details Plasmid MekDD Addgene 15268 See Supplementary Table 2 for more details Plasmid H-RasV12 Addgene 18749 See Supplementary Table 2 for more details Plasmid H2B-EGFP Addgene 21210 See Supplementary Table 2 for more details Plasmid EGFP-BubR1WT This study See Supplementary Table 2 for more details Plasmid EGFP-BubR13A This study See Supplementary Table 2 for more details Plasmid EGFP-Mis12-KARD3D This study See Supplementary Table 2 for more details Plasmid pLEP3_EGFP-BubR1WT This study See Supplementary Table 2 for more details Plasmid pLEP3_EGFP-BubR13A This study See Supplementary Table 2 for more details Primary Antibody anti-pT202/Y204 ERK1/2 [rabbit polyclonal] GeneTex GTX24819 Primary Antibody anti-pS133 CREB (1B6) [mouse monoclonal] Cell Signaling 9196 Primary Antibody anti-histone H3 [rabbit polyclonal] Abcam ab1791 Primary Antibody anti-ERK1/2 [rabbit monoclonal] Cell Signaling 4695 Primary Antibody anti-BubR1 (8G1) [mouse monoclonal] Millipore Sigma MAB3612 1:500 Primary Antibody anti-Hec1 (9G3) [mouse monoclonal] GeneTex GTX70268 2 μg/mL Primary Antibody anti-centromere antibody (ACA) [human polyclonal] Antibodies Inc. 15-235 1:600 Primary Antibody anti-pS55 Hec1 [rabbit purified polyclonal] Supp. .. Ref. 14 See Supplementary Table 3 for more details Nucleic Acid Kits NEBNext Ultra II Directional Library Prep Kit for Illumina New England BioLabs E7760S Nucleic Acid Kits NEBNext Poly(A) mRNA Magnetic Isolation Module New England BioLabs E7490S Nucleic Acid Kits RNeasy mini kit Qiagen 74104 Nucleic Acid Kits iScript cDNA synthesis kit Bio-rad 1708890 Plasmid hTert/mp53DD Addgene 11128 See Supplementary Table 2 for more details Plasmid CCND1/Cdk4R24C Addgene 11129 See Supplementary Table 2 for more details Plasmid c-mycT58A/HRasG12V Addgene 11130 See Supplementary Table 2 for more details Plasmid myr-FLAG-Akt Addgene 15294 See Supplementary Table 2 for more details Plasmid MekDD Addgene 15268 See Supplementary Table 2 for more details Plasmid H-RasV12 Addgene 18749 See Supplementary Table 2 for more details Plasmid H2B-EGFP Addgene 21210 See Supplementary Table 2 for more details Plasmid EGFP-BubR1WT This study See Supplementary Table 2 for more details Plasmid EGFP-BubR13A This study See Supplementary Table 2 for more details Plasmid EGFP-Mis12-KARD3D This study See Supplementary Table 2 for more details Plasmid pLEP3_EGFP-BubR1WT This study See Supplementary Table 2 for more details Plasmid pLEP3_EGFP-BubR13A This study See Supplementary Table 2 for more details Primary Antibody anti-pT202/Y204 ERK1/2 [rabbit polyclonal] GeneTex GTX24819 Primary Antibody anti-pS133 CREB (1B6) [mouse monoclonal] Cell Signaling 9196 Primary Antibody anti-histone H3 [rabbit polyclonal] Abcam ab1791 Primary Antibody anti-ERK1/2 [rabbit monoclonal] Cell Signaling 4695 Primary Antibody anti-BubR1 (8G1) [mouse monoclonal] Millipore Sigma MAB3612 1:500 Primary Antibody anti-Hec1 (9G3) [mouse monoclonal] GeneTex GTX70268 2 μg/mL Primary Antibody anti-centromere antibody (ACA) [human polyclonal] Antibodies Inc. 15-235 1:600 Primary Antibody anti-pS55 Hec1 [rabbit purified polyclonal] Supp.
Article Title: A Notch inhibitor plus Resveratrol induced blockade of autophagy drives glioblastoma cell death by promoting a switch to apoptosis
Article Snippet: Antibodies used in this study: anti-Notch 1, anti-cleaved Notch 1 (NICD), anti-HES 1, anti-HEY 1, anti-LC3B, anti-SQSTM1/p62, anti-Beclin-1, anti-pMAPK 38, anti-MAPK 38, anti-pMAPK44/42, anti-MAPK 44/42, anti-caspase-9, anti-caspase-8, anti-caspase-3 and anti-Bad were acquired from Cell Signaling Technology (Danvers, MA, USA); human anti-β-actin (SC-69879), anti-p27 (SC-53871), anti-cyclin E (SC-247), anti-p21 (SC-6246), anti-cyclinB1 (SC-245), anti-p53 (SC-126), anti-cyclin D1 (SC-8396), anti-Cdk4 (SC-23896), anti-Bcl2 (SC-7382), anti-ATG12 (SC-68884) anti-PARP1 (SC-7150) and Protein A/G PLUS-Agarose were from Santa Cruz Biotechnology (Dallas, TX, USA). .. CDK4R24C expression vector (Plasmid #11129) was from Addgene, GFP-LC3 Expression Vector (#CBA-401) was from Cell Biolabs, Inc. (San Diego, CA). .. Human fetal glial cells SVG p12, human glioblastoma cell lines U87MG and T98G and human breast cancer cells T47D, were purchased from ATCC (Manassas, VA, USA).
Article Title: Hyper-active RAS/MAPK introduces cancer-specific mitotic vulnerabilities
Article Snippet: .. To inactivate tumor suppressor pathways in ARPE19 cells viral particles were generated using two dually encoding plasmids: pBabe- hTERT+p53DD (Addgene plasmid # 11128) and pBabe-cyclinD1+CDK4R24C (Addgene plasmid # 11129), which were gifts from Christopher Counter (3). .. Retroviral particles containing oncogenes were generated from the following donor plasmids: pBabe-myr- HA-AKT, (Addgene plasmid # 15294) or pBabe-Puro-MEK-DD (Addgene plasmid # 15268) which were gifts from William Hahn (15); pWZL-H-RASV12 (Addgene plasmid # 18749), a gift from Scott Lowe (16).
Purification:Article Title: Hyper-active RAS/MAPK introduces cancer-specific mitotic vulnerabilities
Article Snippet: .. Ref. 14 See Supplementary Table 3 for more details Nucleic Acid Kits NEBNext Ultra II Directional Library Prep Kit for Illumina New England BioLabs E7760S Nucleic Acid Kits NEBNext Poly(A) mRNA Magnetic Isolation Module New England BioLabs E7490S Nucleic Acid Kits RNeasy mini kit Qiagen 74104 Nucleic Acid Kits iScript cDNA synthesis kit Bio-rad 1708890 Plasmid hTert/mp53DD Addgene 11128 See Supplementary Table 2 for more details Plasmid CCND1/Cdk4R24C Addgene 11129 See Supplementary Table 2 for more details Plasmid c-mycT58A/HRasG12V Addgene 11130 See Supplementary Table 2 for more details Plasmid myr-FLAG-Akt Addgene 15294 See Supplementary Table 2 for more details Plasmid MekDD Addgene 15268 See Supplementary Table 2 for more details Plasmid H-RasV12 Addgene 18749 See Supplementary Table 2 for more details Plasmid H2B-EGFP Addgene 21210 See Supplementary Table 2 for more details Plasmid EGFP-BubR1WT This study See Supplementary Table 2 for more details Plasmid EGFP-BubR13A This study See Supplementary Table 2 for more details Plasmid EGFP-Mis12-KARD3D This study See Supplementary Table 2 for more details Plasmid pLEP3_EGFP-BubR1WT This study See Supplementary Table 2 for more details Plasmid pLEP3_EGFP-BubR13A This study See Supplementary Table 2 for more details Primary Antibody anti-pT202/Y204 ERK1/2 [rabbit polyclonal] GeneTex GTX24819 Primary Antibody anti-pS133 CREB (1B6) [mouse monoclonal] Cell Signaling 9196 Primary Antibody anti-histone H3 [rabbit polyclonal] Abcam ab1791 Primary Antibody anti-ERK1/2 [rabbit monoclonal] Cell Signaling 4695 Primary Antibody anti-BubR1 (8G1) [mouse monoclonal] Millipore Sigma MAB3612 1:500 Primary Antibody anti-Hec1 (9G3) [mouse monoclonal] GeneTex GTX70268 2 μg/mL Primary Antibody anti-centromere antibody (ACA) [human polyclonal] Antibodies Inc. 15-235 1:600 Primary Antibody anti-pS55 Hec1 [rabbit purified polyclonal] Supp. .. Ref. 14 See Supplementary Table 3 for more details Nucleic Acid Kits NEBNext Ultra II Directional Library Prep Kit for Illumina New England BioLabs E7760S Nucleic Acid Kits NEBNext Poly(A) mRNA Magnetic Isolation Module New England BioLabs E7490S Nucleic Acid Kits RNeasy mini kit Qiagen 74104 Nucleic Acid Kits iScript cDNA synthesis kit Bio-rad 1708890 Plasmid hTert/mp53DD Addgene 11128 See Supplementary Table 2 for more details Plasmid CCND1/Cdk4R24C Addgene 11129 See Supplementary Table 2 for more details Plasmid c-mycT58A/HRasG12V Addgene 11130 See Supplementary Table 2 for more details Plasmid myr-FLAG-Akt Addgene 15294 See Supplementary Table 2 for more details Plasmid MekDD Addgene 15268 See Supplementary Table 2 for more details Plasmid H-RasV12 Addgene 18749 See Supplementary Table 2 for more details Plasmid H2B-EGFP Addgene 21210 See Supplementary Table 2 for more details Plasmid EGFP-BubR1WT This study See Supplementary Table 2 for more details Plasmid EGFP-BubR13A This study See Supplementary Table 2 for more details Plasmid EGFP-Mis12-KARD3D This study See Supplementary Table 2 for more details Plasmid pLEP3_EGFP-BubR1WT This study See Supplementary Table 2 for more details Plasmid pLEP3_EGFP-BubR13A This study See Supplementary Table 2 for more details Primary Antibody anti-pT202/Y204 ERK1/2 [rabbit polyclonal] GeneTex GTX24819 Primary Antibody anti-pS133 CREB (1B6) [mouse monoclonal] Cell Signaling 9196 Primary Antibody anti-histone H3 [rabbit polyclonal] Abcam ab1791 Primary Antibody anti-ERK1/2 [rabbit monoclonal] Cell Signaling 4695 Primary Antibody anti-BubR1 (8G1) [mouse monoclonal] Millipore Sigma MAB3612 1:500 Primary Antibody anti-Hec1 (9G3) [mouse monoclonal] GeneTex GTX70268 2 μg/mL Primary Antibody anti-centromere antibody (ACA) [human polyclonal] Antibodies Inc. 15-235 1:600 Primary Antibody anti-pS55 Hec1 [rabbit purified polyclonal] Supp.
Expressing:Article Title: A Notch inhibitor plus Resveratrol induced blockade of autophagy drives glioblastoma cell death by promoting a switch to apoptosis
Article Snippet: Antibodies used in this study: anti-Notch 1, anti-cleaved Notch 1 (NICD), anti-HES 1, anti-HEY 1, anti-LC3B, anti-SQSTM1/p62, anti-Beclin-1, anti-pMAPK 38, anti-MAPK 38, anti-pMAPK44/42, anti-MAPK 44/42, anti-caspase-9, anti-caspase-8, anti-caspase-3 and anti-Bad were acquired from Cell Signaling Technology (Danvers, MA, USA); human anti-β-actin (SC-69879), anti-p27 (SC-53871), anti-cyclin E (SC-247), anti-p21 (SC-6246), anti-cyclinB1 (SC-245), anti-p53 (SC-126), anti-cyclin D1 (SC-8396), anti-Cdk4 (SC-23896), anti-Bcl2 (SC-7382), anti-ATG12 (SC-68884) anti-PARP1 (SC-7150) and Protein A/G PLUS-Agarose were from Santa Cruz Biotechnology (Dallas, TX, USA). .. CDK4R24C expression vector (Plasmid #11129) was from Addgene, GFP-LC3 Expression Vector (#CBA-401) was from Cell Biolabs, Inc. (San Diego, CA). .. Human fetal glial cells SVG p12, human glioblastoma cell lines U87MG and T98G and human breast cancer cells T47D, were purchased from ATCC (Manassas, VA, USA).
Crocin Bleaching Assay:Article Title: A Notch inhibitor plus Resveratrol induced blockade of autophagy drives glioblastoma cell death by promoting a switch to apoptosis
Article Snippet: Antibodies used in this study: anti-Notch 1, anti-cleaved Notch 1 (NICD), anti-HES 1, anti-HEY 1, anti-LC3B, anti-SQSTM1/p62, anti-Beclin-1, anti-pMAPK 38, anti-MAPK 38, anti-pMAPK44/42, anti-MAPK 44/42, anti-caspase-9, anti-caspase-8, anti-caspase-3 and anti-Bad were acquired from Cell Signaling Technology (Danvers, MA, USA); human anti-β-actin (SC-69879), anti-p27 (SC-53871), anti-cyclin E (SC-247), anti-p21 (SC-6246), anti-cyclinB1 (SC-245), anti-p53 (SC-126), anti-cyclin D1 (SC-8396), anti-Cdk4 (SC-23896), anti-Bcl2 (SC-7382), anti-ATG12 (SC-68884) anti-PARP1 (SC-7150) and Protein A/G PLUS-Agarose were from Santa Cruz Biotechnology (Dallas, TX, USA). .. CDK4R24C expression vector (Plasmid #11129) was from Addgene, GFP-LC3 Expression Vector (#CBA-401) was from Cell Biolabs, Inc. (San Diego, CA). .. Human fetal glial cells SVG p12, human glioblastoma cell lines U87MG and T98G and human breast cancer cells T47D, were purchased from ATCC (Manassas, VA, USA).
Generated:Article Title: Hyper-active RAS/MAPK introduces cancer-specific mitotic vulnerabilities
Article Snippet: .. To inactivate tumor suppressor pathways in ARPE19 cells viral particles were generated using two dually encoding plasmids: pBabe- hTERT+p53DD (Addgene plasmid # 11128) and pBabe-cyclinD1+CDK4R24C (Addgene plasmid # 11129), which were gifts from Christopher Counter (3). .. Retroviral particles containing oncogenes were generated from the following donor plasmids: pBabe-myr- HA-AKT, (Addgene plasmid # 15294) or pBabe-Puro-MEK-DD (Addgene plasmid # 15268) which were gifts from William Hahn (15); pWZL-H-RASV12 (Addgene plasmid # 18749), a gift from Scott Lowe (16).
Article Title: Antagonism among DUX family members evolved from an ancestral toxic single homeodomain protein.
Article Snippet: Control LHCN-M2 immortalized human myoblasts38 and FSHD (M008) immortalized human myoblasts were cultured in F10 medium (HyClone) with 20% FBS (Peak Serum), 2-mercaptoethanol 1x (Gibco), 10-9 M dexamethasone (Sigma), 10 ng/mL bFGF (Peprotech), Glutamax (Gibco), and Penicillin/Streptomycin (P/S, Gibco) at 37 C and 5% CO2. .. Immortalized M008 cells were derived by transducing primary myoblasts obtained from CD56 sorting of mononuclear cells from a muscle biopsy of an individual with FSHD under a protocol approved by the University of Minnesota IRB, with pbabe-cyclinD1+CDK4R24C (gift of Christopher Counter, Addgene)39 and pLV-hTERT-IRES-hygro (gift of TobiasMeyer, Addgene).40 TheM007iDUX4 andM008iDUXA cell lines were generated fromM007 andM008 immortalizedmyoblasts, which were derived by transduction of primary myoblasts from control and FSHD donors respectively, with two lentivectors, FUGW-rtTA and pSAM2-DUXA as previously described.13 The LHCN-M2iDUX4&DUXA cell line was generated by transducing LHCN-M2iDUX4 cells with pSAM-DUXA-ires-GFP lentivirus. .. GFP positive cells were FACS sorted two days post infection using a BD FACSAria (BD Biosciences, Franklin Lakes, NJ).
Transduction:Article Title: Antagonism among DUX family members evolved from an ancestral toxic single homeodomain protein.
Article Snippet: Control LHCN-M2 immortalized human myoblasts38 and FSHD (M008) immortalized human myoblasts were cultured in F10 medium (HyClone) with 20% FBS (Peak Serum), 2-mercaptoethanol 1x (Gibco), 10-9 M dexamethasone (Sigma), 10 ng/mL bFGF (Peprotech), Glutamax (Gibco), and Penicillin/Streptomycin (P/S, Gibco) at 37 C and 5% CO2. .. Immortalized M008 cells were derived by transducing primary myoblasts obtained from CD56 sorting of mononuclear cells from a muscle biopsy of an individual with FSHD under a protocol approved by the University of Minnesota IRB, with pbabe-cyclinD1+CDK4R24C (gift of Christopher Counter, Addgene)39 and pLV-hTERT-IRES-hygro (gift of TobiasMeyer, Addgene).40 TheM007iDUX4 andM008iDUXA cell lines were generated fromM007 andM008 immortalizedmyoblasts, which were derived by transduction of primary myoblasts from control and FSHD donors respectively, with two lentivectors, FUGW-rtTA and pSAM2-DUXA as previously described.13 The LHCN-M2iDUX4&DUXA cell line was generated by transducing LHCN-M2iDUX4 cells with pSAM-DUXA-ires-GFP lentivirus. .. GFP positive cells were FACS sorted two days post infection using a BD FACSAria (BD Biosciences, Franklin Lakes, NJ).
Control:Article Title: Antagonism among DUX family members evolved from an ancestral toxic single homeodomain protein.
Article Snippet: Control LHCN-M2 immortalized human myoblasts38 and FSHD (M008) immortalized human myoblasts were cultured in F10 medium (HyClone) with 20% FBS (Peak Serum), 2-mercaptoethanol 1x (Gibco), 10-9 M dexamethasone (Sigma), 10 ng/mL bFGF (Peprotech), Glutamax (Gibco), and Penicillin/Streptomycin (P/S, Gibco) at 37 C and 5% CO2. .. Immortalized M008 cells were derived by transducing primary myoblasts obtained from CD56 sorting of mononuclear cells from a muscle biopsy of an individual with FSHD under a protocol approved by the University of Minnesota IRB, with pbabe-cyclinD1+CDK4R24C (gift of Christopher Counter, Addgene)39 and pLV-hTERT-IRES-hygro (gift of TobiasMeyer, Addgene).40 TheM007iDUX4 andM008iDUXA cell lines were generated fromM007 andM008 immortalizedmyoblasts, which were derived by transduction of primary myoblasts from control and FSHD donors respectively, with two lentivectors, FUGW-rtTA and pSAM2-DUXA as previously described.13 The LHCN-M2iDUX4&DUXA cell line was generated by transducing LHCN-M2iDUX4 cells with pSAM-DUXA-ires-GFP lentivirus. .. GFP positive cells were FACS sorted two days post infection using a BD FACSAria (BD Biosciences, Franklin Lakes, NJ).
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