The isogenic PCa cell lines LNCaP, C4-2, and C4-2B were cultured in 5% (v/v) heat inactivated fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA) in RPMI 1640 (Gibco, Thermo Fisher Scientific, Waltham Massachusetts) with 1 × penicillin/streptomycin (Gibco) and 1 × L-glutamine (Gibco). Cells were incubated at 37 °C in a humidified 5% (v/v) CO2 atmosphere and passaged at 90-95% confluency with 0.25% (w/v) trypsin-0.38% (w/v) EDTA solution (Gibco) with a 1:10 seeding density. The moderately differentiated ovarian adenocarcinoma SKOV3ip cells were cultured as described previously27 (link).
For shRNA viral induction, C4-2 cells were seeded into a 12-well plate at approximately 40% confluency with normal culturing media for 24 hrs. Media was removed and replaced with culturing media containing 5 µg/mL polybrene. Either control shRNA lentivirus (sc-108080) or SEMA3A directed shRNA lentivirus (sc-36470-V) (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the well and incubated in standard conditions for 24 hours after which the viral media was removed and replaced with full media for an additional 24 hrs. Transduced cells were washed with PBS, detached from the plate with trypsin-EDTA, counted and subjected to the cell-substrate activity assay as described below.
For shRNA viral induction, C4-2 cells were seeded into a 12-well plate at approximately 40% confluency with normal culturing media for 24 hrs. Media was removed and replaced with culturing media containing 5 µg/mL polybrene. Either control shRNA lentivirus (sc-108080) or SEMA3A directed shRNA lentivirus (sc-36470-V) (Santa Cruz Biotechnology, Santa Cruz, CA) was added to the well and incubated in standard conditions for 24 hours after which the viral media was removed and replaced with full media for an additional 24 hrs. Transduced cells were washed with PBS, detached from the plate with trypsin-EDTA, counted and subjected to the cell-substrate activity assay as described below.
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