Pe ampho

The E7 sequences of A1, A4, and A5 were amplified using PCR with primers (forward, 5′-TTG CGG CCG CAC CAT GCA TGG AGA TAC ACC TAC ATT GC-3′; and reverse, 5′-TTG CGG CCG CTG GTT TCT GAG AAC AGA TGG GGC ACA C-3′) from clinical specimens, and cloned into the NotI site of p3xFLAG-CMV14 (Sigma-Aldrich, St. Louis, MO, USA) to fuse the 3xFLAG sequence to the C-terminus of the E7 protein. Then, the E7-FLAG sequence was amplified using PCR, and cloned between the PacI and XhoI sites of a retroviral transfer plasmid, pMXs-puro (Cell Biolabs, San Diego, CA, USA). A retrovirus vector expressing E7-FLAG was prepared via transfection of the transfer plasmid into GP2-293 packaging cells together with an envelope expression plasmid, pE-ampho (Takara). Human cervical keratinocytes immortalized with telomerase reverse transcriptase [36 (link)] were infected with the recombinant retrovirus expressing E7-FLAG, and selected with 1 μg/mL puromycin for 72 h, followed by culture without the drug for 48 h. The surviving cells were pooled and harvested for western blot analyses.

A full-length cDNA for mouse Panx1 (GenBank accession number NM_019482.2) was prepared by RT-PCR from mouse BMDMs, and its authenticity was verified by DNA sequencing. The caspase-recognition sequence, Asp-Ile-Ile-Asp, at amino acid position 375–378 in mouse Panx1 was mutated by recombinant PCR to Ala-Ile-Ile-Ala. The cDNA was Flag-tagged at the C-terminus, and introduced into the pMXs-puro retroviral vector (Kitamura et al., 2003 (link)). Human 293T cells were co-transfected with the pMXs-puro expression vector, together with pEF-gag-pol and pE-Ampho (Takara Bio). 2 days later, the amphotropic retrovirus in the culture medium was concentrated by centrifugation at 6000×g for 16 hr, and used to infect W3 cells in the presence of 10 μg/ml polybrene. Transformants were selected in medium containing 1.0 μg/ml puromycin. To confirm the expression of mouse Panx1, the cells were permeabilized with 0.3% saponin, and stained with FITC-conjugated anti-Flag M2 (Sigma), followed by a flow cytometry with FACSCalibur (BD Bioscience, San Jose, CA).