Detection Assay:Article Title: ATAD2 drives melanoma growth and progression and inhibits ferroptosis.
Article Snippet: Reagents and tools table Reagent or resource Source Identifier Antibodies ACTINB Cell Signaling Cat# 4970 ATAD2 Abcam Cat# ab244431 Phospho-ERK Cell Signaling Cat# 4376 Total-ERK Cell Signaling Cat# 4695 E2F1 Cell Signaling Cat# 3742 CDKN1A Cell Signaling Cat# 2947 Phospho-AMPK Cell Signaling Cat# 2531 Total-AMPK Cell Signaling Cat# 2532 GPX4 Cell Signaling Cat# 52455 V5-tag Cell Signaling Cat# 13202 Chemicals, peptides, and recombinant proteins DMEM GIBCO Cat# 11965-092 RPMI GIBCO Cat# 11875-093 Fetal bovine serum GIBCO Cat# 10437-028 Trypsin-EDTA GIBCO Cat# 25200-056 Penicillin–streptomycin GIBCO Cat# 15140-122 Effectene Transfection Reagent QIAGEN Cat# 301427 Methylthiazole tetrazolium (MTT) Sigma-Aldrich Cat# M5655 Agarose, Low gelling Sigma-Aldrich Cat# A9045 BAY-850 Sigma, MedChem Cat# SML2748, Cat# HY-119254 Trametinib SelleckChem Cat# GSK1120212 Erastin SelleckChem Cat# S7242 Compound C/ Dorsomorphin SelleckChem Cat# S7840 AICAR SelleckChem Cat# S1802 UC2288 SelleckChem Cat# E1190 Reagent or resource Source Identifier Camptothesin SelleckChem Cat# S1288 Human TGF-beta1 recombinant Protein, PeproTech ThermoFisher Scientific Cat# 100-21C-50UG Critical commercial assays In vitro cellular senescence assay Cell Signaling Technology. .. Cat# 25833 Cellular Reactive Oxygen Species Detection Assay Kit Abcam Cat# ab186027 Caspase 3 colorimetric assay kit Sigma Cat# CASP3C CellROX Green flow Cytometry Assay Kit Life Technologies Cat# C10492 CUT&RUN Assay Kit Cell Signaling Cat#86652 Human IL-6 ELISA Kit ThermoFisher Scientific Cat# EH2IL6 Deposited data RNA-Seq performed with A375 and SKMEL103 cells treated with either DMSO or BAY850 This paper GEO: GSE255437 Experimental models: cell lines/organoids/PDXs 293 T ATCC ATCC CRL-3216 A375 ATCC ATCC CRL-1619 M14 CYTION Cat# 302163 SKMEL-103 Sigma Cat# SCC439 SKMEL-2 ATCC ATCC HTB-68 Melanoma PDX Jackson Laboratory TM01386 Primary human fibroblast cells ATCC ATCC PCS-201-012 Experimental models: organisms/strains Mouse: NSG mice Jackson Laboratory Stock No. 005557 shRNAs ATAD2 shRNA Sigma-Aldrich TRC clone ID: TRCN0000158771 E2F1 shRNA Sigma-Aldrich TRC clone ID: TRCN0000000251 Oligonucleotides Forward primer Reverse primer Human ATAD2 AAGGAAGTTGA AACCTACCACCG GCAAGTTGCTC CGTTATTTCCA Human E2F1 ACGCTATGAGA CCTCACTGAA TCCTGGGTCA ACCCCTCAAG Human CDKN1A TGTCCGTCAG AACCCATGC AAAGTCGA AGTTCCATCGCTC Human GPX4 GAGGCAAGACC GAAGTAAACTAC CCGAACTGGT TACACGGGAA Human ACTINB GTCTTCCCCT CCATCGTGGG CCTCTCTTGCTC TGGGCCTC CDKN1A promoter CUT&RUN primers GAGGAGGGA AGTGCCCTCC CGCCGAGCC AGCTGAGCCT © The Author(s) EMBO reports 15 Reagent or resource Source Identifier ATAD2 promoter CUT&RUN primers GAGCGCGCAGAGGCCTCC GAGCTCGGCG GAAGGAGAC ACTINB promoter CUT&RUN primers TCTTGGCTGG GCGTGACTGT AAGGTGGGCT CTACAGGGCA Recombinant DNA Plasmid: piggyBac GFP-Luc Ding et al, 2005 (PMID:16096065) N/A Plasmid: Act-PBase Ding et al, 2005 (PMID:16096065) N/A Plasmid: psPAX2 Addgene Cat#12260, RRID:Addgene_12260 Plasmid: pMD2.G Addgene Cat#12259, RRID:Addgene_12259 Plasmid: GPX4 ORF Horizon discovery Cat#OHS608521383245 Plasmid: GPX4 ORF Origene Cat#RC218291L3 Software and algorithms Prism 10.0 GraphPad www.graphpad.com/ scientific.software/ prism ImageJ https://imagej.nih.gov/ij N/A .. Melanoma cell lines (A375,M14, SKMEL-2, SKMEL-103), HEK-293T and primary human fibroblast cells were purchased from American Type Culture Collection (ATCC), Sigma and Cytion as listed in Reagents and Tools Table and maintained in a humidified atmosphere of 5% CO2 at 37 °C in Dulbecco’s modified Eagle medium (DMEM; Life Technologies, Carlsbad, CA, USA) or Roswell Park Memorial Institute-1640 Medium (RPMI)-1640 Medium; (Life Technologies), each supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (both from Life Technologies).
Colorimetric Assay:Article Title: ATAD2 drives melanoma growth and progression and inhibits ferroptosis.
Article Snippet: Reagents and tools table Reagent or resource Source Identifier Antibodies ACTINB Cell Signaling Cat# 4970 ATAD2 Abcam Cat# ab244431 Phospho-ERK Cell Signaling Cat# 4376 Total-ERK Cell Signaling Cat# 4695 E2F1 Cell Signaling Cat# 3742 CDKN1A Cell Signaling Cat# 2947 Phospho-AMPK Cell Signaling Cat# 2531 Total-AMPK Cell Signaling Cat# 2532 GPX4 Cell Signaling Cat# 52455 V5-tag Cell Signaling Cat# 13202 Chemicals, peptides, and recombinant proteins DMEM GIBCO Cat# 11965-092 RPMI GIBCO Cat# 11875-093 Fetal bovine serum GIBCO Cat# 10437-028 Trypsin-EDTA GIBCO Cat# 25200-056 Penicillin–streptomycin GIBCO Cat# 15140-122 Effectene Transfection Reagent QIAGEN Cat# 301427 Methylthiazole tetrazolium (MTT) Sigma-Aldrich Cat# M5655 Agarose, Low gelling Sigma-Aldrich Cat# A9045 BAY-850 Sigma, MedChem Cat# SML2748, Cat# HY-119254 Trametinib SelleckChem Cat# GSK1120212 Erastin SelleckChem Cat# S7242 Compound C/ Dorsomorphin SelleckChem Cat# S7840 AICAR SelleckChem Cat# S1802 UC2288 SelleckChem Cat# E1190 Reagent or resource Source Identifier Camptothesin SelleckChem Cat# S1288 Human TGF-beta1 recombinant Protein, PeproTech ThermoFisher Scientific Cat# 100-21C-50UG Critical commercial assays In vitro cellular senescence assay Cell Signaling Technology. .. Cat# 25833 Cellular Reactive Oxygen Species Detection Assay Kit Abcam Cat# ab186027 Caspase 3 colorimetric assay kit Sigma Cat# CASP3C CellROX Green flow Cytometry Assay Kit Life Technologies Cat# C10492 CUT&RUN Assay Kit Cell Signaling Cat#86652 Human IL-6 ELISA Kit ThermoFisher Scientific Cat# EH2IL6 Deposited data RNA-Seq performed with A375 and SKMEL103 cells treated with either DMSO or BAY850 This paper GEO: GSE255437 Experimental models: cell lines/organoids/PDXs 293 T ATCC ATCC CRL-3216 A375 ATCC ATCC CRL-1619 M14 CYTION Cat# 302163 SKMEL-103 Sigma Cat# SCC439 SKMEL-2 ATCC ATCC HTB-68 Melanoma PDX Jackson Laboratory TM01386 Primary human fibroblast cells ATCC ATCC PCS-201-012 Experimental models: organisms/strains Mouse: NSG mice Jackson Laboratory Stock No. 005557 shRNAs ATAD2 shRNA Sigma-Aldrich TRC clone ID: TRCN0000158771 E2F1 shRNA Sigma-Aldrich TRC clone ID: TRCN0000000251 Oligonucleotides Forward primer Reverse primer Human ATAD2 AAGGAAGTTGA AACCTACCACCG GCAAGTTGCTC CGTTATTTCCA Human E2F1 ACGCTATGAGA CCTCACTGAA TCCTGGGTCA ACCCCTCAAG Human CDKN1A TGTCCGTCAG AACCCATGC AAAGTCGA AGTTCCATCGCTC Human GPX4 GAGGCAAGACC GAAGTAAACTAC CCGAACTGGT TACACGGGAA Human ACTINB GTCTTCCCCT CCATCGTGGG CCTCTCTTGCTC TGGGCCTC CDKN1A promoter CUT&RUN primers GAGGAGGGA AGTGCCCTCC CGCCGAGCC AGCTGAGCCT © The Author(s) EMBO reports 15 Reagent or resource Source Identifier ATAD2 promoter CUT&RUN primers GAGCGCGCAGAGGCCTCC GAGCTCGGCG GAAGGAGAC ACTINB promoter CUT&RUN primers TCTTGGCTGG GCGTGACTGT AAGGTGGGCT CTACAGGGCA Recombinant DNA Plasmid: piggyBac GFP-Luc Ding et al, 2005 (PMID:16096065) N/A Plasmid: Act-PBase Ding et al, 2005 (PMID:16096065) N/A Plasmid: psPAX2 Addgene Cat#12260, RRID:Addgene_12260 Plasmid: pMD2.G Addgene Cat#12259, RRID:Addgene_12259 Plasmid: GPX4 ORF Horizon discovery Cat#OHS608521383245 Plasmid: GPX4 ORF Origene Cat#RC218291L3 Software and algorithms Prism 10.0 GraphPad www.graphpad.com/ scientific.software/ prism ImageJ https://imagej.nih.gov/ij N/A .. Melanoma cell lines (A375,M14, SKMEL-2, SKMEL-103), HEK-293T and primary human fibroblast cells were purchased from American Type Culture Collection (ATCC), Sigma and Cytion as listed in Reagents and Tools Table and maintained in a humidified atmosphere of 5% CO2 at 37 °C in Dulbecco’s modified Eagle medium (DMEM; Life Technologies, Carlsbad, CA, USA) or Roswell Park Memorial Institute-1640 Medium (RPMI)-1640 Medium; (Life Technologies), each supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (both from Life Technologies).
Flow Cytometry:Article Title: ATAD2 drives melanoma growth and progression and inhibits ferroptosis.
Article Snippet: Reagents and tools table Reagent or resource Source Identifier Antibodies ACTINB Cell Signaling Cat# 4970 ATAD2 Abcam Cat# ab244431 Phospho-ERK Cell Signaling Cat# 4376 Total-ERK Cell Signaling Cat# 4695 E2F1 Cell Signaling Cat# 3742 CDKN1A Cell Signaling Cat# 2947 Phospho-AMPK Cell Signaling Cat# 2531 Total-AMPK Cell Signaling Cat# 2532 GPX4 Cell Signaling Cat# 52455 V5-tag Cell Signaling Cat# 13202 Chemicals, peptides, and recombinant proteins DMEM GIBCO Cat# 11965-092 RPMI GIBCO Cat# 11875-093 Fetal bovine serum GIBCO Cat# 10437-028 Trypsin-EDTA GIBCO Cat# 25200-056 Penicillin–streptomycin GIBCO Cat# 15140-122 Effectene Transfection Reagent QIAGEN Cat# 301427 Methylthiazole tetrazolium (MTT) Sigma-Aldrich Cat# M5655 Agarose, Low gelling Sigma-Aldrich Cat# A9045 BAY-850 Sigma, MedChem Cat# SML2748, Cat# HY-119254 Trametinib SelleckChem Cat# GSK1120212 Erastin SelleckChem Cat# S7242 Compound C/ Dorsomorphin SelleckChem Cat# S7840 AICAR SelleckChem Cat# S1802 UC2288 SelleckChem Cat# E1190 Reagent or resource Source Identifier Camptothesin SelleckChem Cat# S1288 Human TGF-beta1 recombinant Protein, PeproTech ThermoFisher Scientific Cat# 100-21C-50UG Critical commercial assays In vitro cellular senescence assay Cell Signaling Technology. .. Cat# 25833 Cellular Reactive Oxygen Species Detection Assay Kit Abcam Cat# ab186027 Caspase 3 colorimetric assay kit Sigma Cat# CASP3C CellROX Green flow Cytometry Assay Kit Life Technologies Cat# C10492 CUT&RUN Assay Kit Cell Signaling Cat#86652 Human IL-6 ELISA Kit ThermoFisher Scientific Cat# EH2IL6 Deposited data RNA-Seq performed with A375 and SKMEL103 cells treated with either DMSO or BAY850 This paper GEO: GSE255437 Experimental models: cell lines/organoids/PDXs 293 T ATCC ATCC CRL-3216 A375 ATCC ATCC CRL-1619 M14 CYTION Cat# 302163 SKMEL-103 Sigma Cat# SCC439 SKMEL-2 ATCC ATCC HTB-68 Melanoma PDX Jackson Laboratory TM01386 Primary human fibroblast cells ATCC ATCC PCS-201-012 Experimental models: organisms/strains Mouse: NSG mice Jackson Laboratory Stock No. 005557 shRNAs ATAD2 shRNA Sigma-Aldrich TRC clone ID: TRCN0000158771 E2F1 shRNA Sigma-Aldrich TRC clone ID: TRCN0000000251 Oligonucleotides Forward primer Reverse primer Human ATAD2 AAGGAAGTTGA AACCTACCACCG GCAAGTTGCTC CGTTATTTCCA Human E2F1 ACGCTATGAGA CCTCACTGAA TCCTGGGTCA ACCCCTCAAG Human CDKN1A TGTCCGTCAG AACCCATGC AAAGTCGA AGTTCCATCGCTC Human GPX4 GAGGCAAGACC GAAGTAAACTAC CCGAACTGGT TACACGGGAA Human ACTINB GTCTTCCCCT CCATCGTGGG CCTCTCTTGCTC TGGGCCTC CDKN1A promoter CUT&RUN primers GAGGAGGGA AGTGCCCTCC CGCCGAGCC AGCTGAGCCT © The Author(s) EMBO reports 15 Reagent or resource Source Identifier ATAD2 promoter CUT&RUN primers GAGCGCGCAGAGGCCTCC GAGCTCGGCG GAAGGAGAC ACTINB promoter CUT&RUN primers TCTTGGCTGG GCGTGACTGT AAGGTGGGCT CTACAGGGCA Recombinant DNA Plasmid: piggyBac GFP-Luc Ding et al, 2005 (PMID:16096065) N/A Plasmid: Act-PBase Ding et al, 2005 (PMID:16096065) N/A Plasmid: psPAX2 Addgene Cat#12260, RRID:Addgene_12260 Plasmid: pMD2.G Addgene Cat#12259, RRID:Addgene_12259 Plasmid: GPX4 ORF Horizon discovery Cat#OHS608521383245 Plasmid: GPX4 ORF Origene Cat#RC218291L3 Software and algorithms Prism 10.0 GraphPad www.graphpad.com/ scientific.software/ prism ImageJ https://imagej.nih.gov/ij N/A .. Melanoma cell lines (A375,M14, SKMEL-2, SKMEL-103), HEK-293T and primary human fibroblast cells were purchased from American Type Culture Collection (ATCC), Sigma and Cytion as listed in Reagents and Tools Table and maintained in a humidified atmosphere of 5% CO2 at 37 °C in Dulbecco’s modified Eagle medium (DMEM; Life Technologies, Carlsbad, CA, USA) or Roswell Park Memorial Institute-1640 Medium (RPMI)-1640 Medium; (Life Technologies), each supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (both from Life Technologies).
Enzyme-linked Immunosorbent Assay:Article Title: ATAD2 drives melanoma growth and progression and inhibits ferroptosis.
Article Snippet: Reagents and tools table Reagent or resource Source Identifier Antibodies ACTINB Cell Signaling Cat# 4970 ATAD2 Abcam Cat# ab244431 Phospho-ERK Cell Signaling Cat# 4376 Total-ERK Cell Signaling Cat# 4695 E2F1 Cell Signaling Cat# 3742 CDKN1A Cell Signaling Cat# 2947 Phospho-AMPK Cell Signaling Cat# 2531 Total-AMPK Cell Signaling Cat# 2532 GPX4 Cell Signaling Cat# 52455 V5-tag Cell Signaling Cat# 13202 Chemicals, peptides, and recombinant proteins DMEM GIBCO Cat# 11965-092 RPMI GIBCO Cat# 11875-093 Fetal bovine serum GIBCO Cat# 10437-028 Trypsin-EDTA GIBCO Cat# 25200-056 Penicillin–streptomycin GIBCO Cat# 15140-122 Effectene Transfection Reagent QIAGEN Cat# 301427 Methylthiazole tetrazolium (MTT) Sigma-Aldrich Cat# M5655 Agarose, Low gelling Sigma-Aldrich Cat# A9045 BAY-850 Sigma, MedChem Cat# SML2748, Cat# HY-119254 Trametinib SelleckChem Cat# GSK1120212 Erastin SelleckChem Cat# S7242 Compound C/ Dorsomorphin SelleckChem Cat# S7840 AICAR SelleckChem Cat# S1802 UC2288 SelleckChem Cat# E1190 Reagent or resource Source Identifier Camptothesin SelleckChem Cat# S1288 Human TGF-beta1 recombinant Protein, PeproTech ThermoFisher Scientific Cat# 100-21C-50UG Critical commercial assays In vitro cellular senescence assay Cell Signaling Technology. .. Cat# 25833 Cellular Reactive Oxygen Species Detection Assay Kit Abcam Cat# ab186027 Caspase 3 colorimetric assay kit Sigma Cat# CASP3C CellROX Green flow Cytometry Assay Kit Life Technologies Cat# C10492 CUT&RUN Assay Kit Cell Signaling Cat#86652 Human IL-6 ELISA Kit ThermoFisher Scientific Cat# EH2IL6 Deposited data RNA-Seq performed with A375 and SKMEL103 cells treated with either DMSO or BAY850 This paper GEO: GSE255437 Experimental models: cell lines/organoids/PDXs 293 T ATCC ATCC CRL-3216 A375 ATCC ATCC CRL-1619 M14 CYTION Cat# 302163 SKMEL-103 Sigma Cat# SCC439 SKMEL-2 ATCC ATCC HTB-68 Melanoma PDX Jackson Laboratory TM01386 Primary human fibroblast cells ATCC ATCC PCS-201-012 Experimental models: organisms/strains Mouse: NSG mice Jackson Laboratory Stock No. 005557 shRNAs ATAD2 shRNA Sigma-Aldrich TRC clone ID: TRCN0000158771 E2F1 shRNA Sigma-Aldrich TRC clone ID: TRCN0000000251 Oligonucleotides Forward primer Reverse primer Human ATAD2 AAGGAAGTTGA AACCTACCACCG GCAAGTTGCTC CGTTATTTCCA Human E2F1 ACGCTATGAGA CCTCACTGAA TCCTGGGTCA ACCCCTCAAG Human CDKN1A TGTCCGTCAG AACCCATGC AAAGTCGA AGTTCCATCGCTC Human GPX4 GAGGCAAGACC GAAGTAAACTAC CCGAACTGGT TACACGGGAA Human ACTINB GTCTTCCCCT CCATCGTGGG CCTCTCTTGCTC TGGGCCTC CDKN1A promoter CUT&RUN primers GAGGAGGGA AGTGCCCTCC CGCCGAGCC AGCTGAGCCT © The Author(s) EMBO reports 15 Reagent or resource Source Identifier ATAD2 promoter CUT&RUN primers GAGCGCGCAGAGGCCTCC GAGCTCGGCG GAAGGAGAC ACTINB promoter CUT&RUN primers TCTTGGCTGG GCGTGACTGT AAGGTGGGCT CTACAGGGCA Recombinant DNA Plasmid: piggyBac GFP-Luc Ding et al, 2005 (PMID:16096065) N/A Plasmid: Act-PBase Ding et al, 2005 (PMID:16096065) N/A Plasmid: psPAX2 Addgene Cat#12260, RRID:Addgene_12260 Plasmid: pMD2.G Addgene Cat#12259, RRID:Addgene_12259 Plasmid: GPX4 ORF Horizon discovery Cat#OHS608521383245 Plasmid: GPX4 ORF Origene Cat#RC218291L3 Software and algorithms Prism 10.0 GraphPad www.graphpad.com/ scientific.software/ prism ImageJ https://imagej.nih.gov/ij N/A .. Melanoma cell lines (A375,M14, SKMEL-2, SKMEL-103), HEK-293T and primary human fibroblast cells were purchased from American Type Culture Collection (ATCC), Sigma and Cytion as listed in Reagents and Tools Table and maintained in a humidified atmosphere of 5% CO2 at 37 °C in Dulbecco’s modified Eagle medium (DMEM; Life Technologies, Carlsbad, CA, USA) or Roswell Park Memorial Institute-1640 Medium (RPMI)-1640 Medium; (Life Technologies), each supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (both from Life Technologies).
RNA sequencing:Article Title: ATAD2 drives melanoma growth and progression and inhibits ferroptosis.
Article Snippet: Reagents and tools table Reagent or resource Source Identifier Antibodies ACTINB Cell Signaling Cat# 4970 ATAD2 Abcam Cat# ab244431 Phospho-ERK Cell Signaling Cat# 4376 Total-ERK Cell Signaling Cat# 4695 E2F1 Cell Signaling Cat# 3742 CDKN1A Cell Signaling Cat# 2947 Phospho-AMPK Cell Signaling Cat# 2531 Total-AMPK Cell Signaling Cat# 2532 GPX4 Cell Signaling Cat# 52455 V5-tag Cell Signaling Cat# 13202 Chemicals, peptides, and recombinant proteins DMEM GIBCO Cat# 11965-092 RPMI GIBCO Cat# 11875-093 Fetal bovine serum GIBCO Cat# 10437-028 Trypsin-EDTA GIBCO Cat# 25200-056 Penicillin–streptomycin GIBCO Cat# 15140-122 Effectene Transfection Reagent QIAGEN Cat# 301427 Methylthiazole tetrazolium (MTT) Sigma-Aldrich Cat# M5655 Agarose, Low gelling Sigma-Aldrich Cat# A9045 BAY-850 Sigma, MedChem Cat# SML2748, Cat# HY-119254 Trametinib SelleckChem Cat# GSK1120212 Erastin SelleckChem Cat# S7242 Compound C/ Dorsomorphin SelleckChem Cat# S7840 AICAR SelleckChem Cat# S1802 UC2288 SelleckChem Cat# E1190 Reagent or resource Source Identifier Camptothesin SelleckChem Cat# S1288 Human TGF-beta1 recombinant Protein, PeproTech ThermoFisher Scientific Cat# 100-21C-50UG Critical commercial assays In vitro cellular senescence assay Cell Signaling Technology. .. Cat# 25833 Cellular Reactive Oxygen Species Detection Assay Kit Abcam Cat# ab186027 Caspase 3 colorimetric assay kit Sigma Cat# CASP3C CellROX Green flow Cytometry Assay Kit Life Technologies Cat# C10492 CUT&RUN Assay Kit Cell Signaling Cat#86652 Human IL-6 ELISA Kit ThermoFisher Scientific Cat# EH2IL6 Deposited data RNA-Seq performed with A375 and SKMEL103 cells treated with either DMSO or BAY850 This paper GEO: GSE255437 Experimental models: cell lines/organoids/PDXs 293 T ATCC ATCC CRL-3216 A375 ATCC ATCC CRL-1619 M14 CYTION Cat# 302163 SKMEL-103 Sigma Cat# SCC439 SKMEL-2 ATCC ATCC HTB-68 Melanoma PDX Jackson Laboratory TM01386 Primary human fibroblast cells ATCC ATCC PCS-201-012 Experimental models: organisms/strains Mouse: NSG mice Jackson Laboratory Stock No. 005557 shRNAs ATAD2 shRNA Sigma-Aldrich TRC clone ID: TRCN0000158771 E2F1 shRNA Sigma-Aldrich TRC clone ID: TRCN0000000251 Oligonucleotides Forward primer Reverse primer Human ATAD2 AAGGAAGTTGA AACCTACCACCG GCAAGTTGCTC CGTTATTTCCA Human E2F1 ACGCTATGAGA CCTCACTGAA TCCTGGGTCA ACCCCTCAAG Human CDKN1A TGTCCGTCAG AACCCATGC AAAGTCGA AGTTCCATCGCTC Human GPX4 GAGGCAAGACC GAAGTAAACTAC CCGAACTGGT TACACGGGAA Human ACTINB GTCTTCCCCT CCATCGTGGG CCTCTCTTGCTC TGGGCCTC CDKN1A promoter CUT&RUN primers GAGGAGGGA AGTGCCCTCC CGCCGAGCC AGCTGAGCCT © The Author(s) EMBO reports 15 Reagent or resource Source Identifier ATAD2 promoter CUT&RUN primers GAGCGCGCAGAGGCCTCC GAGCTCGGCG GAAGGAGAC ACTINB promoter CUT&RUN primers TCTTGGCTGG GCGTGACTGT AAGGTGGGCT CTACAGGGCA Recombinant DNA Plasmid: piggyBac GFP-Luc Ding et al, 2005 (PMID:16096065) N/A Plasmid: Act-PBase Ding et al, 2005 (PMID:16096065) N/A Plasmid: psPAX2 Addgene Cat#12260, RRID:Addgene_12260 Plasmid: pMD2.G Addgene Cat#12259, RRID:Addgene_12259 Plasmid: GPX4 ORF Horizon discovery Cat#OHS608521383245 Plasmid: GPX4 ORF Origene Cat#RC218291L3 Software and algorithms Prism 10.0 GraphPad www.graphpad.com/ scientific.software/ prism ImageJ https://imagej.nih.gov/ij N/A .. Melanoma cell lines (A375,M14, SKMEL-2, SKMEL-103), HEK-293T and primary human fibroblast cells were purchased from American Type Culture Collection (ATCC), Sigma and Cytion as listed in Reagents and Tools Table and maintained in a humidified atmosphere of 5% CO2 at 37 °C in Dulbecco’s modified Eagle medium (DMEM; Life Technologies, Carlsbad, CA, USA) or Roswell Park Memorial Institute-1640 Medium (RPMI)-1640 Medium; (Life Technologies), each supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (both from Life Technologies).
shRNA:Article Title: ATAD2 drives melanoma growth and progression and inhibits ferroptosis.
Article Snippet: Reagents and tools table Reagent or resource Source Identifier Antibodies ACTINB Cell Signaling Cat# 4970 ATAD2 Abcam Cat# ab244431 Phospho-ERK Cell Signaling Cat# 4376 Total-ERK Cell Signaling Cat# 4695 E2F1 Cell Signaling Cat# 3742 CDKN1A Cell Signaling Cat# 2947 Phospho-AMPK Cell Signaling Cat# 2531 Total-AMPK Cell Signaling Cat# 2532 GPX4 Cell Signaling Cat# 52455 V5-tag Cell Signaling Cat# 13202 Chemicals, peptides, and recombinant proteins DMEM GIBCO Cat# 11965-092 RPMI GIBCO Cat# 11875-093 Fetal bovine serum GIBCO Cat# 10437-028 Trypsin-EDTA GIBCO Cat# 25200-056 Penicillin–streptomycin GIBCO Cat# 15140-122 Effectene Transfection Reagent QIAGEN Cat# 301427 Methylthiazole tetrazolium (MTT) Sigma-Aldrich Cat# M5655 Agarose, Low gelling Sigma-Aldrich Cat# A9045 BAY-850 Sigma, MedChem Cat# SML2748, Cat# HY-119254 Trametinib SelleckChem Cat# GSK1120212 Erastin SelleckChem Cat# S7242 Compound C/ Dorsomorphin SelleckChem Cat# S7840 AICAR SelleckChem Cat# S1802 UC2288 SelleckChem Cat# E1190 Reagent or resource Source Identifier Camptothesin SelleckChem Cat# S1288 Human TGF-beta1 recombinant Protein, PeproTech ThermoFisher Scientific Cat# 100-21C-50UG Critical commercial assays In vitro cellular senescence assay Cell Signaling Technology. .. Cat# 25833 Cellular Reactive Oxygen Species Detection Assay Kit Abcam Cat# ab186027 Caspase 3 colorimetric assay kit Sigma Cat# CASP3C CellROX Green flow Cytometry Assay Kit Life Technologies Cat# C10492 CUT&RUN Assay Kit Cell Signaling Cat#86652 Human IL-6 ELISA Kit ThermoFisher Scientific Cat# EH2IL6 Deposited data RNA-Seq performed with A375 and SKMEL103 cells treated with either DMSO or BAY850 This paper GEO: GSE255437 Experimental models: cell lines/organoids/PDXs 293 T ATCC ATCC CRL-3216 A375 ATCC ATCC CRL-1619 M14 CYTION Cat# 302163 SKMEL-103 Sigma Cat# SCC439 SKMEL-2 ATCC ATCC HTB-68 Melanoma PDX Jackson Laboratory TM01386 Primary human fibroblast cells ATCC ATCC PCS-201-012 Experimental models: organisms/strains Mouse: NSG mice Jackson Laboratory Stock No. 005557 shRNAs ATAD2 shRNA Sigma-Aldrich TRC clone ID: TRCN0000158771 E2F1 shRNA Sigma-Aldrich TRC clone ID: TRCN0000000251 Oligonucleotides Forward primer Reverse primer Human ATAD2 AAGGAAGTTGA AACCTACCACCG GCAAGTTGCTC CGTTATTTCCA Human E2F1 ACGCTATGAGA CCTCACTGAA TCCTGGGTCA ACCCCTCAAG Human CDKN1A TGTCCGTCAG AACCCATGC AAAGTCGA AGTTCCATCGCTC Human GPX4 GAGGCAAGACC GAAGTAAACTAC CCGAACTGGT TACACGGGAA Human ACTINB GTCTTCCCCT CCATCGTGGG CCTCTCTTGCTC TGGGCCTC CDKN1A promoter CUT&RUN primers GAGGAGGGA AGTGCCCTCC CGCCGAGCC AGCTGAGCCT © The Author(s) EMBO reports 15 Reagent or resource Source Identifier ATAD2 promoter CUT&RUN primers GAGCGCGCAGAGGCCTCC GAGCTCGGCG GAAGGAGAC ACTINB promoter CUT&RUN primers TCTTGGCTGG GCGTGACTGT AAGGTGGGCT CTACAGGGCA Recombinant DNA Plasmid: piggyBac GFP-Luc Ding et al, 2005 (PMID:16096065) N/A Plasmid: Act-PBase Ding et al, 2005 (PMID:16096065) N/A Plasmid: psPAX2 Addgene Cat#12260, RRID:Addgene_12260 Plasmid: pMD2.G Addgene Cat#12259, RRID:Addgene_12259 Plasmid: GPX4 ORF Horizon discovery Cat#OHS608521383245 Plasmid: GPX4 ORF Origene Cat#RC218291L3 Software and algorithms Prism 10.0 GraphPad www.graphpad.com/ scientific.software/ prism ImageJ https://imagej.nih.gov/ij N/A .. Melanoma cell lines (A375,M14, SKMEL-2, SKMEL-103), HEK-293T and primary human fibroblast cells were purchased from American Type Culture Collection (ATCC), Sigma and Cytion as listed in Reagents and Tools Table and maintained in a humidified atmosphere of 5% CO2 at 37 °C in Dulbecco’s modified Eagle medium (DMEM; Life Technologies, Carlsbad, CA, USA) or Roswell Park Memorial Institute-1640 Medium (RPMI)-1640 Medium; (Life Technologies), each supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (both from Life Technologies).
Recombinant:Article Title: ATAD2 drives melanoma growth and progression and inhibits ferroptosis.
Article Snippet: Reagents and tools table Reagent or resource Source Identifier Antibodies ACTINB Cell Signaling Cat# 4970 ATAD2 Abcam Cat# ab244431 Phospho-ERK Cell Signaling Cat# 4376 Total-ERK Cell Signaling Cat# 4695 E2F1 Cell Signaling Cat# 3742 CDKN1A Cell Signaling Cat# 2947 Phospho-AMPK Cell Signaling Cat# 2531 Total-AMPK Cell Signaling Cat# 2532 GPX4 Cell Signaling Cat# 52455 V5-tag Cell Signaling Cat# 13202 Chemicals, peptides, and recombinant proteins DMEM GIBCO Cat# 11965-092 RPMI GIBCO Cat# 11875-093 Fetal bovine serum GIBCO Cat# 10437-028 Trypsin-EDTA GIBCO Cat# 25200-056 Penicillin–streptomycin GIBCO Cat# 15140-122 Effectene Transfection Reagent QIAGEN Cat# 301427 Methylthiazole tetrazolium (MTT) Sigma-Aldrich Cat# M5655 Agarose, Low gelling Sigma-Aldrich Cat# A9045 BAY-850 Sigma, MedChem Cat# SML2748, Cat# HY-119254 Trametinib SelleckChem Cat# GSK1120212 Erastin SelleckChem Cat# S7242 Compound C/ Dorsomorphin SelleckChem Cat# S7840 AICAR SelleckChem Cat# S1802 UC2288 SelleckChem Cat# E1190 Reagent or resource Source Identifier Camptothesin SelleckChem Cat# S1288 Human TGF-beta1 recombinant Protein, PeproTech ThermoFisher Scientific Cat# 100-21C-50UG Critical commercial assays In vitro cellular senescence assay Cell Signaling Technology. .. Cat# 25833 Cellular Reactive Oxygen Species Detection Assay Kit Abcam Cat# ab186027 Caspase 3 colorimetric assay kit Sigma Cat# CASP3C CellROX Green flow Cytometry Assay Kit Life Technologies Cat# C10492 CUT&RUN Assay Kit Cell Signaling Cat#86652 Human IL-6 ELISA Kit ThermoFisher Scientific Cat# EH2IL6 Deposited data RNA-Seq performed with A375 and SKMEL103 cells treated with either DMSO or BAY850 This paper GEO: GSE255437 Experimental models: cell lines/organoids/PDXs 293 T ATCC ATCC CRL-3216 A375 ATCC ATCC CRL-1619 M14 CYTION Cat# 302163 SKMEL-103 Sigma Cat# SCC439 SKMEL-2 ATCC ATCC HTB-68 Melanoma PDX Jackson Laboratory TM01386 Primary human fibroblast cells ATCC ATCC PCS-201-012 Experimental models: organisms/strains Mouse: NSG mice Jackson Laboratory Stock No. 005557 shRNAs ATAD2 shRNA Sigma-Aldrich TRC clone ID: TRCN0000158771 E2F1 shRNA Sigma-Aldrich TRC clone ID: TRCN0000000251 Oligonucleotides Forward primer Reverse primer Human ATAD2 AAGGAAGTTGA AACCTACCACCG GCAAGTTGCTC CGTTATTTCCA Human E2F1 ACGCTATGAGA CCTCACTGAA TCCTGGGTCA ACCCCTCAAG Human CDKN1A TGTCCGTCAG AACCCATGC AAAGTCGA AGTTCCATCGCTC Human GPX4 GAGGCAAGACC GAAGTAAACTAC CCGAACTGGT TACACGGGAA Human ACTINB GTCTTCCCCT CCATCGTGGG CCTCTCTTGCTC TGGGCCTC CDKN1A promoter CUT&RUN primers GAGGAGGGA AGTGCCCTCC CGCCGAGCC AGCTGAGCCT © The Author(s) EMBO reports 15 Reagent or resource Source Identifier ATAD2 promoter CUT&RUN primers GAGCGCGCAGAGGCCTCC GAGCTCGGCG GAAGGAGAC ACTINB promoter CUT&RUN primers TCTTGGCTGG GCGTGACTGT AAGGTGGGCT CTACAGGGCA Recombinant DNA Plasmid: piggyBac GFP-Luc Ding et al, 2005 (PMID:16096065) N/A Plasmid: Act-PBase Ding et al, 2005 (PMID:16096065) N/A Plasmid: psPAX2 Addgene Cat#12260, RRID:Addgene_12260 Plasmid: pMD2.G Addgene Cat#12259, RRID:Addgene_12259 Plasmid: GPX4 ORF Horizon discovery Cat#OHS608521383245 Plasmid: GPX4 ORF Origene Cat#RC218291L3 Software and algorithms Prism 10.0 GraphPad www.graphpad.com/ scientific.software/ prism ImageJ https://imagej.nih.gov/ij N/A .. Melanoma cell lines (A375,M14, SKMEL-2, SKMEL-103), HEK-293T and primary human fibroblast cells were purchased from American Type Culture Collection (ATCC), Sigma and Cytion as listed in Reagents and Tools Table and maintained in a humidified atmosphere of 5% CO2 at 37 °C in Dulbecco’s modified Eagle medium (DMEM; Life Technologies, Carlsbad, CA, USA) or Roswell Park Memorial Institute-1640 Medium (RPMI)-1640 Medium; (Life Technologies), each supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (both from Life Technologies).
Plasmid Preparation:Article Title: ATAD2 drives melanoma growth and progression and inhibits ferroptosis.
Article Snippet: Reagents and tools table Reagent or resource Source Identifier Antibodies ACTINB Cell Signaling Cat# 4970 ATAD2 Abcam Cat# ab244431 Phospho-ERK Cell Signaling Cat# 4376 Total-ERK Cell Signaling Cat# 4695 E2F1 Cell Signaling Cat# 3742 CDKN1A Cell Signaling Cat# 2947 Phospho-AMPK Cell Signaling Cat# 2531 Total-AMPK Cell Signaling Cat# 2532 GPX4 Cell Signaling Cat# 52455 V5-tag Cell Signaling Cat# 13202 Chemicals, peptides, and recombinant proteins DMEM GIBCO Cat# 11965-092 RPMI GIBCO Cat# 11875-093 Fetal bovine serum GIBCO Cat# 10437-028 Trypsin-EDTA GIBCO Cat# 25200-056 Penicillin–streptomycin GIBCO Cat# 15140-122 Effectene Transfection Reagent QIAGEN Cat# 301427 Methylthiazole tetrazolium (MTT) Sigma-Aldrich Cat# M5655 Agarose, Low gelling Sigma-Aldrich Cat# A9045 BAY-850 Sigma, MedChem Cat# SML2748, Cat# HY-119254 Trametinib SelleckChem Cat# GSK1120212 Erastin SelleckChem Cat# S7242 Compound C/ Dorsomorphin SelleckChem Cat# S7840 AICAR SelleckChem Cat# S1802 UC2288 SelleckChem Cat# E1190 Reagent or resource Source Identifier Camptothesin SelleckChem Cat# S1288 Human TGF-beta1 recombinant Protein, PeproTech ThermoFisher Scientific Cat# 100-21C-50UG Critical commercial assays In vitro cellular senescence assay Cell Signaling Technology. .. Cat# 25833 Cellular Reactive Oxygen Species Detection Assay Kit Abcam Cat# ab186027 Caspase 3 colorimetric assay kit Sigma Cat# CASP3C CellROX Green flow Cytometry Assay Kit Life Technologies Cat# C10492 CUT&RUN Assay Kit Cell Signaling Cat#86652 Human IL-6 ELISA Kit ThermoFisher Scientific Cat# EH2IL6 Deposited data RNA-Seq performed with A375 and SKMEL103 cells treated with either DMSO or BAY850 This paper GEO: GSE255437 Experimental models: cell lines/organoids/PDXs 293 T ATCC ATCC CRL-3216 A375 ATCC ATCC CRL-1619 M14 CYTION Cat# 302163 SKMEL-103 Sigma Cat# SCC439 SKMEL-2 ATCC ATCC HTB-68 Melanoma PDX Jackson Laboratory TM01386 Primary human fibroblast cells ATCC ATCC PCS-201-012 Experimental models: organisms/strains Mouse: NSG mice Jackson Laboratory Stock No. 005557 shRNAs ATAD2 shRNA Sigma-Aldrich TRC clone ID: TRCN0000158771 E2F1 shRNA Sigma-Aldrich TRC clone ID: TRCN0000000251 Oligonucleotides Forward primer Reverse primer Human ATAD2 AAGGAAGTTGA AACCTACCACCG GCAAGTTGCTC CGTTATTTCCA Human E2F1 ACGCTATGAGA CCTCACTGAA TCCTGGGTCA ACCCCTCAAG Human CDKN1A TGTCCGTCAG AACCCATGC AAAGTCGA AGTTCCATCGCTC Human GPX4 GAGGCAAGACC GAAGTAAACTAC CCGAACTGGT TACACGGGAA Human ACTINB GTCTTCCCCT CCATCGTGGG CCTCTCTTGCTC TGGGCCTC CDKN1A promoter CUT&RUN primers GAGGAGGGA AGTGCCCTCC CGCCGAGCC AGCTGAGCCT © The Author(s) EMBO reports 15 Reagent or resource Source Identifier ATAD2 promoter CUT&RUN primers GAGCGCGCAGAGGCCTCC GAGCTCGGCG GAAGGAGAC ACTINB promoter CUT&RUN primers TCTTGGCTGG GCGTGACTGT AAGGTGGGCT CTACAGGGCA Recombinant DNA Plasmid: piggyBac GFP-Luc Ding et al, 2005 (PMID:16096065) N/A Plasmid: Act-PBase Ding et al, 2005 (PMID:16096065) N/A Plasmid: psPAX2 Addgene Cat#12260, RRID:Addgene_12260 Plasmid: pMD2.G Addgene Cat#12259, RRID:Addgene_12259 Plasmid: GPX4 ORF Horizon discovery Cat#OHS608521383245 Plasmid: GPX4 ORF Origene Cat#RC218291L3 Software and algorithms Prism 10.0 GraphPad www.graphpad.com/ scientific.software/ prism ImageJ https://imagej.nih.gov/ij N/A .. Melanoma cell lines (A375,M14, SKMEL-2, SKMEL-103), HEK-293T and primary human fibroblast cells were purchased from American Type Culture Collection (ATCC), Sigma and Cytion as listed in Reagents and Tools Table and maintained in a humidified atmosphere of 5% CO2 at 37 °C in Dulbecco’s modified Eagle medium (DMEM; Life Technologies, Carlsbad, CA, USA) or Roswell Park Memorial Institute-1640 Medium (RPMI)-1640 Medium; (Life Technologies), each supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (both from Life Technologies).
Software:Article Title: ATAD2 drives melanoma growth and progression and inhibits ferroptosis.
Article Snippet: Reagents and tools table Reagent or resource Source Identifier Antibodies ACTINB Cell Signaling Cat# 4970 ATAD2 Abcam Cat# ab244431 Phospho-ERK Cell Signaling Cat# 4376 Total-ERK Cell Signaling Cat# 4695 E2F1 Cell Signaling Cat# 3742 CDKN1A Cell Signaling Cat# 2947 Phospho-AMPK Cell Signaling Cat# 2531 Total-AMPK Cell Signaling Cat# 2532 GPX4 Cell Signaling Cat# 52455 V5-tag Cell Signaling Cat# 13202 Chemicals, peptides, and recombinant proteins DMEM GIBCO Cat# 11965-092 RPMI GIBCO Cat# 11875-093 Fetal bovine serum GIBCO Cat# 10437-028 Trypsin-EDTA GIBCO Cat# 25200-056 Penicillin–streptomycin GIBCO Cat# 15140-122 Effectene Transfection Reagent QIAGEN Cat# 301427 Methylthiazole tetrazolium (MTT) Sigma-Aldrich Cat# M5655 Agarose, Low gelling Sigma-Aldrich Cat# A9045 BAY-850 Sigma, MedChem Cat# SML2748, Cat# HY-119254 Trametinib SelleckChem Cat# GSK1120212 Erastin SelleckChem Cat# S7242 Compound C/ Dorsomorphin SelleckChem Cat# S7840 AICAR SelleckChem Cat# S1802 UC2288 SelleckChem Cat# E1190 Reagent or resource Source Identifier Camptothesin SelleckChem Cat# S1288 Human TGF-beta1 recombinant Protein, PeproTech ThermoFisher Scientific Cat# 100-21C-50UG Critical commercial assays In vitro cellular senescence assay Cell Signaling Technology. .. Cat# 25833 Cellular Reactive Oxygen Species Detection Assay Kit Abcam Cat# ab186027 Caspase 3 colorimetric assay kit Sigma Cat# CASP3C CellROX Green flow Cytometry Assay Kit Life Technologies Cat# C10492 CUT&RUN Assay Kit Cell Signaling Cat#86652 Human IL-6 ELISA Kit ThermoFisher Scientific Cat# EH2IL6 Deposited data RNA-Seq performed with A375 and SKMEL103 cells treated with either DMSO or BAY850 This paper GEO: GSE255437 Experimental models: cell lines/organoids/PDXs 293 T ATCC ATCC CRL-3216 A375 ATCC ATCC CRL-1619 M14 CYTION Cat# 302163 SKMEL-103 Sigma Cat# SCC439 SKMEL-2 ATCC ATCC HTB-68 Melanoma PDX Jackson Laboratory TM01386 Primary human fibroblast cells ATCC ATCC PCS-201-012 Experimental models: organisms/strains Mouse: NSG mice Jackson Laboratory Stock No. 005557 shRNAs ATAD2 shRNA Sigma-Aldrich TRC clone ID: TRCN0000158771 E2F1 shRNA Sigma-Aldrich TRC clone ID: TRCN0000000251 Oligonucleotides Forward primer Reverse primer Human ATAD2 AAGGAAGTTGA AACCTACCACCG GCAAGTTGCTC CGTTATTTCCA Human E2F1 ACGCTATGAGA CCTCACTGAA TCCTGGGTCA ACCCCTCAAG Human CDKN1A TGTCCGTCAG AACCCATGC AAAGTCGA AGTTCCATCGCTC Human GPX4 GAGGCAAGACC GAAGTAAACTAC CCGAACTGGT TACACGGGAA Human ACTINB GTCTTCCCCT CCATCGTGGG CCTCTCTTGCTC TGGGCCTC CDKN1A promoter CUT&RUN primers GAGGAGGGA AGTGCCCTCC CGCCGAGCC AGCTGAGCCT © The Author(s) EMBO reports 15 Reagent or resource Source Identifier ATAD2 promoter CUT&RUN primers GAGCGCGCAGAGGCCTCC GAGCTCGGCG GAAGGAGAC ACTINB promoter CUT&RUN primers TCTTGGCTGG GCGTGACTGT AAGGTGGGCT CTACAGGGCA Recombinant DNA Plasmid: piggyBac GFP-Luc Ding et al, 2005 (PMID:16096065) N/A Plasmid: Act-PBase Ding et al, 2005 (PMID:16096065) N/A Plasmid: psPAX2 Addgene Cat#12260, RRID:Addgene_12260 Plasmid: pMD2.G Addgene Cat#12259, RRID:Addgene_12259 Plasmid: GPX4 ORF Horizon discovery Cat#OHS608521383245 Plasmid: GPX4 ORF Origene Cat#RC218291L3 Software and algorithms Prism 10.0 GraphPad www.graphpad.com/ scientific.software/ prism ImageJ https://imagej.nih.gov/ij N/A .. Melanoma cell lines (A375,M14, SKMEL-2, SKMEL-103), HEK-293T and primary human fibroblast cells were purchased from American Type Culture Collection (ATCC), Sigma and Cytion as listed in Reagents and Tools Table and maintained in a humidified atmosphere of 5% CO2 at 37 °C in Dulbecco’s modified Eagle medium (DMEM; Life Technologies, Carlsbad, CA, USA) or Roswell Park Memorial Institute-1640 Medium (RPMI)-1640 Medium; (Life Technologies), each supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (both from Life Technologies).
MTT Assay:Article Title: Phytochemical characterization, total phenolic and flavonoid content, antioxidant capacity, enzymatic profiling, and cytotoxicity of Bidens pilosa and Croton sp. from Colombia for applications in skin health
Article Snippet: .. Cytotoxicity was assessed in HaCaT keratinocytes (cat. no. 300493, CLS Cell Lines Service GmbH, Germany) and A375 melanoma cells (cat. no. 300110, CLS Cell Lines Service LLC, USA) using the colorimetric MTT assay, adapted with minor modifications from Caballero-Gallardo et al. [ ]. .. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin and seeded in 96-well plates at a density of 1.5 × 104 cells per well.
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