siRNAs against G6PD were On-target smart pools from Dharmacon and were transfected using RNAiMax (Invitrogen) according to the manufacturer’s protocol. Cells were analyzed 72 h after transfection.
On target smart pools
Manufactured by Horizon Discovery
On-target smart pools are a collection of pre-designed and validated CRISPR guide RNAs (crRNAs) that are optimized to target a specific gene. The smart pools are designed to maximize on-target editing efficiency while minimizing off-target effects.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Rnaimax, by Thermo Fisher Scientific (1 mentions)
Celltiter glo assay kit, by Promega (1 mentions)
Plasmotest, by InvivoGen (1 mentions)
Celltiter glo kit, by Promega (1 mentions)
Non targeting control sirna, by Horizon Discovery (1 mentions)
Unc3230, by MedChemExpress (1 mentions)
Lipofectamine crisprmax, by Thermo Fisher Scientific (1 mentions)
5 protocols using on target smart pools
1
Silencing G6PD Gene Expression
2
Plasmid and siRNA Manipulation Protocol
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pCMV6-TUSC3, pCMV6-NM23H1, and pCMV6-Synoviolin (HRD1)-Flag plasmids were purchased from Origene. pcDNA-IRE1α-GFP, pcDNA-TP53-GFP, pcDNA-Ubiquitin-HA plasmids were purchased from Addgene Company. For the TUSC3, the cDNAs were subcloned into pcDNA4.1-V5-His vector with EcoR I and Xho I restriction sites or pcDNA3-HA vector with Hind III restriction site. The 3’UTR of TUSC3-1 and TUSC3-2 plasmids were purchased from Origene and the 3’UTRs were subcloned into pGL3 Vectors with Xho I restriction site. On-Target Smart Pools containing 4 different siRNAs on each target gene of TUSC3, TP53, HRD1, ATF6α or NM23H1 were purchased from Dharmacon company. The primer sets for gene manipulation are shown in Supplementary Tables 1 .
3
Cell Line Authentication and Viability Assays
4
Synthesis and Screening of Triazine Hydrazones
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1 H-indole-3-carbaldehyde (4-anilino-6-[4-morpholinyl]-1,3,5-triazin-2-yl)hydrazone (WX8) was from SPECS (MLS000543798). UNC3230 was from MedChemExpress (HY-110150). ONTARGET SMART-pools and non-targeting control siRNAs were designed by Dharmacon. Cell-Titer Glo kit was from Promega Biotechnology (G7570).
5
Genetic Perturbation in Breast Cell Lines
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MCF10A and MDA-MB-231 knockdown cells were obtained by transfecting 20 nM siRNAs with Lipofectamine RNAiMAX (Invitrogen). siRNAs were Dharmacon ON-TARGET SMART Pools (L-010259-00-0010 for PPP2R1A, L-032698-00-0010 for NHSL1, L-010640-00-0010 for NCKAP1) or Sigma siRNAs CCTTTGTACGCTTTGCTCA and GCCACTACAACAGAATTTT to target RAC1. Cells were analyzed after 3 days.
MCF10A knockout cell lines were generated with CRISPR/Cas9 system. The following gRNAs were used:
PPP2R1A 5′-CATAGACGAACTCCGCAATG-3′
NHSL1 5′-TCGGCTTTCCTCATCTAGGT-3′
non-targeting 5′-AAAUGUGAGAUCAGAGUAAU-3′.
Cells were transfected with gRNA, tracrRNA, and Cas9 protein by Lipofectamine CRISPRMAX™ (all reagents from ThermoFischer Scientific). After 2 days, cells were diluted at 0.8 cells/well in 96-well plates. Single clones were expanded and analyzed by western blot. The positive clones were confirmed by sequencing.
MCF10A knockout cell lines were generated with CRISPR/Cas9 system. The following gRNAs were used:
PPP2R1A 5′-CATAGACGAACTCCGCAATG-3′
NHSL1 5′-TCGGCTTTCCTCATCTAGGT-3′
non-targeting 5′-AAAUGUGAGAUCAGAGUAAU-3′.
Cells were transfected with gRNA, tracrRNA, and Cas9 protein by Lipofectamine CRISPRMAX™ (all reagents from ThermoFischer Scientific). After 2 days, cells were diluted at 0.8 cells/well in 96-well plates. Single clones were expanded and analyzed by western blot. The positive clones were confirmed by sequencing.
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