Stock solutions (10 mM) of trametinib, ABT-199, ABT-263, AT-101, sabutoclax, and UMI-77 (Selleckchem, Houston, TX, USA) were prepared in DMSO and stored at −80 °C. Appropriate concentrations of each drug were prepared in DMSO prior to use. Cell lines were transfected with siRNAs to BCL-2 (ON-TARGETplus Human BCL2 SMARTpool; Dharmacon, Lafayette, CO, USA), BCL-XL (Hs_BCL2L1_2, QIAGEN, Hilden, Germany), MCL-1 (Hs_MCL1_6, QIAGEN), BIM (Hs_BCL2L11_5, QIAGEN), and BAD (Hs_BAD_3, QIAGEN) using 4D-Nucleofector system (Lonza, Basel, Switzerland). AllStars Negative Control siRNA (QIAGEN) was the negative control.
The BIM-GFP expression plasmid was constructed by cloning the human BIMEL cDNA (BCL2L11-001, ENST00000393256) into a pMCEF vector and moving the BamHI and AgeI restriction fragment into the pEGFP-N1 vector. The plasmid was transfected into SD1 cells using 4D-Nucleofector system (Lonza) and GFP+ cells were sorted by flow cytometry after 48 h.
The BIM-GFP expression plasmid was constructed by cloning the human BIMEL cDNA (BCL2L11-001, ENST00000393256) into a pMCEF vector and moving the BamHI and AgeI restriction fragment into the pEGFP-N1 vector. The plasmid was transfected into SD1 cells using 4D-Nucleofector system (Lonza) and GFP+ cells were sorted by flow cytometry after 48 h.