Psuper retro puro vector plasmid

The S100A10 shRNA1 knockdown construct was designed by cloning the following dsRNA oligo (Table S11) into the pSUPER‐retro‐puro vector plasmid (OligoEngine, Seattle, WA, USA). To establish stable S100A10 knockdown cell lines, Phoenix cells were first transfected with 4 μg of the pSUPER‐retro scramble control and S100A10 shRNA1 plasmids using with lipofectamine 2000 transfection reagent (Cat no. 11668019, Invitrogen, Burlington, ON, Canada). Panc‐1 cells were transduced with the retroviral supernatants and puromycin selection started at 48 h posttransduction. The pBabe‐puro control (#1764) and KRAS^G12D (#58902) constructs were obtained the plasmid depository Addgene (Cambridge, MA, USA). The transfected clones were selected in 1 μg·mL⁻¹ puromycin.

Either mouse Zip13-shRNA or non-specific (Scramble) DNA (Supplementary Table S1) were inserted into the pSUPER.retro.puro vector plasmid (OligoEngine, Seattle, WA, USA) according to the manufacturer’s instructions to generate Zip13-shRNA or Scramble plasmids. C2C12 cells were transfected with either Zip13-shRNA or Scramble plasmids using Lipofectamine LTX (Thermo Fisher Scientific) according to the manufacturer’s instructions and cultured in a medium containing 5 µg/mL puromycin for 48 h. Cells were then collected, and the KD efficiency of Zip13 mRNA was examined by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis. The remaining cells were seeded by limiting dilution to generate a monoclonal cell line for both Zip13-KD and Scramble C2C12 cells. We examined Zip13 mRNA expression in the twelve monoclonal cell lines and selected two clones (clone 6 and 7) based on the KD efficiency of Zip13, and four clones of Scramble C2C12 cells (clone number: 1, 5, 9, and 13) were used as the experimental control.