Human bladder cancer cells (UM-UC-1, UM-UC-3) were purchased from ATCC, and authenticity was confirmed by short tandem repeat (STR) analysis20 (link) or by MSK-IMPACT analysis72 (link). All cell lines are routinely screened for mycoplasma. Cells were cultured in Minimal Essential Medium (UM-UC-1, UM-UC-3) supplemented with 10% FBS. Western blotting was performed as described previously20 (link). Primary antibodies were used as follows: FOXA1 (1:500, ab23738, Abcam), PD-L1 (1:1000, ab213524, abcam), GAPDH (14C10) (1:1000, #2118, Cell signaling).
To establish UM-UC-1 FOXA1 knockout (KO), UM-UC-1 (2 × 105) cells were transfected with 2.5 mg of HNF-3alpha CRISPR/Cas9 KO plasmid (Santa Cruz, sc-400743) using lipofectamine3000 (Thermo fisher scientific). Three GFP-positive cells were sorted in a single well of 96-well plate containing 100 ml of medium by Aria II cell sorter (BD Biosciences). Sorted cells were expanded and knockout of FOXA1 in UM-UC-1 cells was confirmed by western blot analysis.
To establish UM-UC-1 FOXA1 knockout (KO), UM-UC-1 (2 × 105) cells were transfected with 2.5 mg of HNF-3alpha CRISPR/Cas9 KO plasmid (Santa Cruz, sc-400743) using lipofectamine3000 (Thermo fisher scientific). Three GFP-positive cells were sorted in a single well of 96-well plate containing 100 ml of medium by Aria II cell sorter (BD Biosciences). Sorted cells were expanded and knockout of FOXA1 in UM-UC-1 cells was confirmed by western blot analysis.
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