Knock-Out:Article Title: Oncogenic PIK3CA corrupts growth factor signaling specificity.
Article Snippet: .. Reagents and tools table Reagent/resource Reference or source Identifier or catalog number Experimental models Human cervical cancer (HeLa) cells ATCC #CCL-2 Immortalized mouse embryonic fibroblasts (MEFs) with wild-type PIK3CA and PIK3CA knock-out Foukas et al, 2010 Human lung adenocarcinoma A549 cells ATCC #CCL-185 WTC11 line (RRID: CVCL_Y803) with wildtype PIK3CA, PIK3CAWT/ H1047R or PIK3CAH1047R/H1047R Madsen et al, 2019 Parental line: RRID: CVCL_Y803 Recombinant DNA pSBbi-FoxO1_1R_10A_3D (designated MB40) Addgene RRID:Addgene_106278 pCMV(CAT)T7-SB100 (designated MB43) Addgene RRID:Addgene_34879 pNES-EGFP-C1-PHARNO(I303E)x2 Addgene RRID:Addgene_116868 pNES-mCherry-C1-TAPP1cPHx3 Addgene RRID:Addgene_116855 PH-BTK-GFP (original) Addgene RRID:Addgene_51463 pNES-mCherry-C1-PHmutARNO-R280A-2GI303Ex2 MRC Reagents & Services #DU78205 pNES-EGFP-C1-PHmutARNO-R280A-2GI303Ex2 MRC Reagents & Services #DU78206 © The Author(s) Molecular Systems Biology 11 D ow nloaded from https://w w w .em bopress.org on D ecem ber 25, 2024 from IP 2402:800:6343:b0f8:400f:1124:9738:8c7d. .. Reagent/resource Reference or source Identifier or catalog number pNES-EGFP-C1-PH-BTKWT MRC Reagents & Services #DU78207 pNES-mCherry-C1-PHBTK-MUT MRC Reagents & Services #DU78208 pNES-EGFP-C1-PH-AKT2WT MRC Reagents & Services #DU78209 pNES-mCherry-C1-PHAKT2-MUT MRC Reagents & Services #DU78210 pNES-GFP-PH-TH-BTK MRC Reagents & Services #DU78211 pNES-GFP-PHmut-TH-BTK MRC Reagents & Services #DU78212 pUC19 New England Biolabs #09052008 Antibodies Primary antibodies for Western blotting See Appendix Table S1 Secondary antibodies for Western blotting See Appendix Table S2 Primary antibodies for mass cytometry See Appendix Table S3 Oligonucleotides and other sequence-based reagents HDR001 Madsen et al, 2019 and this study HDR002 Madsen et al, 2019 and this study sgRNA for PIK3CA exon 21 Madsen et al, 2019 and this study Synthego EZ sgRNA custom order PIK3CA exon 21 Sanger sequencing primers Madsen et al, 2019 and this study Sigma Aldrich oligonucleotide custom order MiSeq PIK3CA exon 21 primers This study Sigma Aldrich oligonucleotide custom order BTK PH domain cloning and site-directed mutagenesis primers This study Sigma Aldrich oligonucleotide custom order AKT2 PH domain cloning and site-directed mutagenesis primers This study Sigma Aldrich oligonucleotide custom order RT-qPCR primers See Appendix Table S4 Chemicals, enzymes, and other reagents NuncTM Cell-Culture Treated Multidishes Thermo Fisher Scientific #140675 Corning 25 cm2 Rectangular Canted Neck Cell Culture Flask with Vent Cap Corning #430639 DMEM high glucose (with 4 mM L-Glutamine and 1 mM sodium pyruvate) Thermo Fisher Scientific #41966-029 L-Glutamine Sigma Aldrich #G7513 10% fetal bovine serum Pan-Biotech #P30-8500 RPMI-1640 with GlutaMax and Sodium Bicarbonate Thermo Fisher Scientific #61870-036 Reagent/resource Reference or source Identifier or catalog number Sodium Pyruvate Thermo Fisher Scientific #11360-039 DPBS Sigma Aldrich #RNBH8966 DPBS Thermo Fisher Scientific #14190-094 TrypLETM Express Enzyme Thermo Fisher Scientific #12605028 or #1260421 DMEM/F12 Thermo Fisher Scientific #21331-046 Essential 8 Flex Medium Thermo Fisher Scientific #A2858501 Cultrex Stem Cell Qualified Reduced Growth Factor Basement Membrane R&D Systems #3434-010-02 10 μM Y-27632 dihydrochloride Bio-Techne #1254/10 StemPro Accutase Thermo Fisher Scientific #A1110501 Alt-RTM S.p.
Recombinant:Article Title: Oncogenic PIK3CA corrupts growth factor signaling specificity.
Article Snippet: .. Reagents and tools table Reagent/resource Reference or source Identifier or catalog number Experimental models Human cervical cancer (HeLa) cells ATCC #CCL-2 Immortalized mouse embryonic fibroblasts (MEFs) with wild-type PIK3CA and PIK3CA knock-out Foukas et al, 2010 Human lung adenocarcinoma A549 cells ATCC #CCL-185 WTC11 line (RRID: CVCL_Y803) with wildtype PIK3CA, PIK3CAWT/ H1047R or PIK3CAH1047R/H1047R Madsen et al, 2019 Parental line: RRID: CVCL_Y803 Recombinant DNA pSBbi-FoxO1_1R_10A_3D (designated MB40) Addgene RRID:Addgene_106278 pCMV(CAT)T7-SB100 (designated MB43) Addgene RRID:Addgene_34879 pNES-EGFP-C1-PHARNO(I303E)x2 Addgene RRID:Addgene_116868 pNES-mCherry-C1-TAPP1cPHx3 Addgene RRID:Addgene_116855 PH-BTK-GFP (original) Addgene RRID:Addgene_51463 pNES-mCherry-C1-PHmutARNO-R280A-2GI303Ex2 MRC Reagents & Services #DU78205 pNES-EGFP-C1-PHmutARNO-R280A-2GI303Ex2 MRC Reagents & Services #DU78206 © The Author(s) Molecular Systems Biology 11 D ow nloaded from https://w w w .em bopress.org on D ecem ber 25, 2024 from IP 2402:800:6343:b0f8:400f:1124:9738:8c7d. .. Reagent/resource Reference or source Identifier or catalog number pNES-EGFP-C1-PH-BTKWT MRC Reagents & Services #DU78207 pNES-mCherry-C1-PHBTK-MUT MRC Reagents & Services #DU78208 pNES-EGFP-C1-PH-AKT2WT MRC Reagents & Services #DU78209 pNES-mCherry-C1-PHAKT2-MUT MRC Reagents & Services #DU78210 pNES-GFP-PH-TH-BTK MRC Reagents & Services #DU78211 pNES-GFP-PHmut-TH-BTK MRC Reagents & Services #DU78212 pUC19 New England Biolabs #09052008 Antibodies Primary antibodies for Western blotting See Appendix Table S1 Secondary antibodies for Western blotting See Appendix Table S2 Primary antibodies for mass cytometry See Appendix Table S3 Oligonucleotides and other sequence-based reagents HDR001 Madsen et al, 2019 and this study HDR002 Madsen et al, 2019 and this study sgRNA for PIK3CA exon 21 Madsen et al, 2019 and this study Synthego EZ sgRNA custom order PIK3CA exon 21 Sanger sequencing primers Madsen et al, 2019 and this study Sigma Aldrich oligonucleotide custom order MiSeq PIK3CA exon 21 primers This study Sigma Aldrich oligonucleotide custom order BTK PH domain cloning and site-directed mutagenesis primers This study Sigma Aldrich oligonucleotide custom order AKT2 PH domain cloning and site-directed mutagenesis primers This study Sigma Aldrich oligonucleotide custom order RT-qPCR primers See Appendix Table S4 Chemicals, enzymes, and other reagents NuncTM Cell-Culture Treated Multidishes Thermo Fisher Scientific #140675 Corning 25 cm2 Rectangular Canted Neck Cell Culture Flask with Vent Cap Corning #430639 DMEM high glucose (with 4 mM L-Glutamine and 1 mM sodium pyruvate) Thermo Fisher Scientific #41966-029 L-Glutamine Sigma Aldrich #G7513 10% fetal bovine serum Pan-Biotech #P30-8500 RPMI-1640 with GlutaMax and Sodium Bicarbonate Thermo Fisher Scientific #61870-036 Reagent/resource Reference or source Identifier or catalog number Sodium Pyruvate Thermo Fisher Scientific #11360-039 DPBS Sigma Aldrich #RNBH8966 DPBS Thermo Fisher Scientific #14190-094 TrypLETM Express Enzyme Thermo Fisher Scientific #12605028 or #1260421 DMEM/F12 Thermo Fisher Scientific #21331-046 Essential 8 Flex Medium Thermo Fisher Scientific #A2858501 Cultrex Stem Cell Qualified Reduced Growth Factor Basement Membrane R&D Systems #3434-010-02 10 μM Y-27632 dihydrochloride Bio-Techne #1254/10 StemPro Accutase Thermo Fisher Scientific #A1110501 Alt-RTM S.p.
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