Shrna lentiviral particles

Knockdown of CA XII expression in T47D cells has also been previously described [72 ]. Briefly, cells were transfected with short hairpin RNA (shRNA) lentiviral particles obtained from Thermo Fisher Scientific (Waltham, MA) against CA XII. Cells were seeded at 5x10⁴ cells/well in 24-well plates and grown for 24 h. Then, cells were infected with lentivirus in serum-free medium for 6 h. The cells were further incubated with normal growth medium for 24 h. GFP expression was monitored to confirm the efficiency of transduction. Stable cells were established by puromycin (2 μg/mL) selection (Sigma Aldrich, St. Louis, MO). CA XII knockdown was confirmed by western blotting.

Knockdown of CAIX and CAXII expression in UFH-001 and T47D cells was performed by transfection of short hairpin RNA (shRNA) lentiviral particles (Thermo scientific) against CAIX and CAXII. In brief, breast cancer cells were seeded at 5x10⁴ cells/well in 24-well plates and grown for 24 hours. Then, cells were infected with lentivirus in serum-free medium for 6 hours. The shRNA target sequences for CAIX and CAXII are shown in S1. The cells were further incubated with normal growth medium for 24 h. GFP expression was monitored to confirm the efficiency of transduction. Stable cells were established by puromycin (2 μg/mL) selection (Sigma Aldrich #P-7255). CAIX and CAXII knockdown was confirmed by western blotting.