Pbabe krasg12d

pBabe-Kras^G12D (#58902) and LT3GEPIR (#111177) were from Addgene. The insert expressing the shRNA was cloned into the vector LT3GEPIR in the XhoI and EcoRI sites. The shRNAs target sequences were selected either from the library described by Feldmann et al.¹⁰⁹ or from the splashRNA database¹¹⁰ (http://splashrna.mskcc.org/). The hairpin targeting sequences are: shLPCAT3 #1 5′-GCCTCTCAATTGCTTATTTTA-3′; shLPCAT3 #2 5′-AAGGAAAGAGAAGTTAAA-3′; shPLA2G4C #1 5′-CAGAATGAATGTGATAGTTCA-3′; shPLA2G4C #2 5′- ACATGGTTATCTCTAAGCAAA-3′; shGPX4 #1 5′-GTGGATGAAGATCCAACCCAA-3′; shGPX4 #2 5′-AGGCAAGACCGAAGTAAACTA-3′. shKRAS #10 5′-AAGTTGAGACCTTCTTAATTGGT-3′; shKRAS #40 5′-TCAGGACTTAGCAAGAAGTTA-3′. Production of lentiviruses was performed as previously described^{12 (link)}.

The plasmids pBabe-puro (Addgene # 1764) were a gift from Hartmut Land and Jay Morgenstern, pBabe-puro HRAS V12 (Addgene # 39526), were a gift from Julian Downward and HRAS V12 (Addgene # 9051) was a gift from William Hahn, pbabe - KRAS G12D (Addgene # 58902) was a gift from Channing Der pBabe-puro HRAS V12 S35 (Addgene # 18746) was a gift from Jay Morgenstern, pBabe-puro HRAS V12 C40 (Addgene # 18747) and pBabe-puro HRAS V12 G37 (Addgene # 18745) was a gift from Scott Lowe S100A10 siRNA was purchased as a pre-designed sequence from Ambion (4392420). The pKRAS-gRNA1-px459-V2 plasmid was made by cloning the annealed oligos: 5′- CAC CGA ATA TAA ACT TGT GGT AGT-3′ and 5′-AAA CAC TAC CAC AAG TTT ATA TTC-3′ into px459-V2 from Addgene (62988) and the pKRAS-gRNA2-px459-V2 was made by cloning the annealed oligos: 5′-CAC CGA AAC TTG TGG TAG TTG GAG C-3′ and 5′-AAA CGC TCC AAC TAC CAC AAG TTT C-3′ into px459-V2 from Addgene. The pGL4.22- S100A10-promoter luciferase reporter plasmid was constructed by cutting the S100A10-promoter from the pCAT-S100A10-promoter plasmid and cloning it into the Kpn I/Sac I restriction sites of pGL4.22 vector (Promega).

Cells (1 × 10⁵) were seeded in a 6-well plate and incubated at 37 °C for 24 h. Plasmids were transfected with TurboFect transfection reagent (Fermentas, Hanover, MD) following the manufacturer’s protocol. pBabe-KrasG12D was purchased from Addgene (#58902).