Panx1 ko crispr cas9 plasmids

The HeLa cell line was a kind gift from Prof. Shin Yonehara. The cell line was authenticated by short-tandem repeat profiling and was checked for mycoplasma infection. The cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, 1 g/L glucose; Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich). The SW480 cell line was obtained from American Type Culture Collection. The cells were cultured in Leibovitz’s L-15 medium (Wako Pure Chemicals, Japan) supplemented with 10% FBS. Apoptosis of HeLa cells was initiated either by adding anti-FAS antibody (125 ng/mL) and CHX (10 µM) or by adding staurosporine (1 µM). Apoptosis of SW480 cells was initiated by adding Trail (50 ng/mL) and anti-His tag antibody (1 µg/mL). Knockout of PANX1 gene was carried out using pre-designed PANX1-KO CRISPR-Cas9 plasmids (Santa Cruz Biotechnology). Briefly, cells were transfected with CRISPR-Cas9 plasmids using PEI-Max (Polysciences Inc) as described previously (Morciano et al., 2020 (link)). After 2 days, each individual cell with strong GFP fluorescence was sorted into a well in 96-well plates using a cell sorter (SH800S, Sony), and then was cultured. Knockout of PANX1 gene in each cultured line was verified by western blotting and by sequencing of the targeted region of the genomic DNA.