Trcn0000059773

Lentivirus expressing short hairpin RNA (shRNA) targeting Cx43 (Sigma, TRCN0000059773) was used to downregulate Cx43 in CTRL1 hiPSC-CFs (Cx43-sh-RNA CFs). Briefly, one day after seeding 60000 cells/12-well, hiPSC-CFs were transduced with viral particles at MOI 1 in fresh FGM3 with 8 μg/mL polybrene overnight. GFP expressing virus (pLV-CMV-GFP) was used as a control (scramble shRNA). 72 h post-transduction, when control cells expressed GFP, infected cells were selected with 1 μg/mL puromycin (Sigma, P7255). After 3 days, remaining cells were fixed for immunofluorescence staining, collected for RNA extraction and dissociated to prepare MTs.

Prostate cancer cell lines LNCaP, C4-2, DU145, PC-3 were maintained in RPMI medium supplemented with 10% fetal bovine serum (FBS), 50 U/ml of penicillin, 50 μg/ml of streptomycin and 2 mM L-glutamine (Life Technologies). LNCaP was purchased from ATCC, C4-2 was kindly provided by Dr. Alex Almasan (Cleveland Clinic), and DU145 and PC-3 were kindly provided by Dr. Nima Sharifi (Cleveland Clinic). LNCaP and PC-3 cells with down-regulated Cx43 were prepared by transduction with lentivirus expressing Cx43 specific shRNA (Sigma, TRCN0000059773 and TRCN0000059775). Lentivirus was produced in 293T cells using standardized protocol with packaging plasmids as described previously [33 (link)], and viral particles containing conditioned medium was filtered through 0.45 μM PVDF membrane and directly used to infect prostate cancer cell lines in the presence of 8 μg/ml polybrene (Sigma). Twenty four hours after infection, cells were selected with puromycin (Sigma) at a final concentration of 3 μg/mL for PC-3 cells and 1 μg/mL for LNCaP cells. Surviving cells were pooled together and maintained in medium containing 1 μg/mL puromycin for both cell lines. Control cells were obtained in a similar manner with lentivirus expressing non-targeting shRNA (Sigma, SHC002).