HO-1 knockout using CRISPR/Cas9

A HO-1 knock out cell line was created using the pSpCas9 BB-2A-GFP (Genescript, USA) vector, containing a single guide RNA (sgRNA) sequence (5’-TGGAGCGTCCGCAACCCGAC-3’), targeting HO-1. Transfections using Lipofectamine 2000 (Invitrogen, USA) were performed according to the manufacturer’s instructions. Cells expressing high GFP were selected by single cell sorting into a 96-well plate containing media with antibiotics. Western blots for HO-1 were used to confirm knockout clones. Parent Capan-1 and HO-1 knockout cells were treated with different concentrations of PL and cultured for 48 h under hypoxia (4% oxygen) in a hypoxia chamber (Heracell VIOS 160i Tri-Gas CO2, Fisher, USA). Cell viability was measured by MTT assay (Promega, USA) as per manufacturer protocol.