BCL-XL-KO MC38 and Renca cells were generated by transient transfection of Cas9 protein-guide RNA complex into parental cells, followed by single clone section and immunoblot to confirm KO of BCL-XL following the instruction (Synthego). The guide RNA used for BCL-XL KO is 5′-AUACUUUUGUGGAACUCUAU-3′.
Cells were plated in 96-well plates at a density of 3 × 103 to 5 × 103 cells per well and cultured overnight. Cells were treated with increasing concentrations of DT2216, PZ15227, ABT199, a BCL2 specific inhibitor10 (link) (LC laboratories), A1155463, a BCL-XL specific inhibitor38 (link) (Selleck Chemicals, Houston, TX), ABT263, a BCL-2/BCL-XL dual inhibitor9 (link) (Selleck Chemicals) or S63845, a MCL-1 selective inhibitor12 (link) (Selleck Chemicals) for 72 h. Cell viability was measured by tetrazolium-based MTS assay as described previously16 (link). Briefly, MTS reagent (Promega, Madison, WI) was supplemented with phenazine methosulfate (Sigma-Aldrich, St. Louis, MO) at a 20:1 ratio and 20 µl was added to each well and incubated for 4 h at 37 °C. Absorbance was measured at 490 nm using a Synergy Neo2 multimode plate reader (Biotek, Winooski, VT) and cell viability was determined for each well. The data were expressed as the average percent of viable cells and fitted in non-linear regression curves using Prism software (GraphPad Software).
Cells were plated in 96-well plates at a density of 3 × 103 to 5 × 103 cells per well and cultured overnight. Cells were treated with increasing concentrations of DT2216, PZ15227, ABT199, a BCL2 specific inhibitor10 (link) (LC laboratories), A1155463, a BCL-XL specific inhibitor38 (link) (Selleck Chemicals, Houston, TX), ABT263, a BCL-2/BCL-XL dual inhibitor9 (link) (Selleck Chemicals) or S63845, a MCL-1 selective inhibitor12 (link) (Selleck Chemicals) for 72 h. Cell viability was measured by tetrazolium-based MTS assay as described previously16 (link). Briefly, MTS reagent (Promega, Madison, WI) was supplemented with phenazine methosulfate (Sigma-Aldrich, St. Louis, MO) at a 20:1 ratio and 20 µl was added to each well and incubated for 4 h at 37 °C. Absorbance was measured at 490 nm using a Synergy Neo2 multimode plate reader (Biotek, Winooski, VT) and cell viability was determined for each well. The data were expressed as the average percent of viable cells and fitted in non-linear regression curves using Prism software (GraphPad Software).
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