All siRNA were purchased from Dharmacon (Lafayette, CO, USA). Untreated cells and cells treated with nontargeted scrambled siRNA (Dharmacon, Catalog Item D-001810-10-20) or luciferase siRNA (Dharmacon, Catalog Item P-002099-01-50) were used as controls. Isoform-specific siRNAs were custom designed using Thermo Scientific siRNA Design Center (Thermo Scientific, Rockford, IL, USA). siRNA specific sequences were 5′-CAUGCUGUUCCAGUGCUCC-3′ for Chk-α, 5′-GAGGAAGACCUGAAGGUUCAGCAUA-3′ for PD-L1 #1, and 5′-CCUACUGGCAUUUGCUGAACGCAUU-3′ for PD-L1 #2.
Cells at 40 to 50% confluency were transfected with 100 nM of scrambled or luciferase siRNA, and with 50 nM or 100 nM of Chk-α- or PD-L1-specific siRNA for individual treatments. For combination siRNA treatments, 50 nM of each specific siRNA was used. Cells were treated with siRNA for 48 h because this incubation period resulted in the most effective downregulation of the target genes. D-FECT 4 (Dharmacon, Catalog Item T-2004-03) was used as the transfection agent for MDA-MB-231, Pa09C and Pa20C cells, and Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA, Catalog Item 11668019) for SUM-149 cells. All transfections were carried out based on established protocols [34 (link)].
Cells at 40 to 50% confluency were transfected with 100 nM of scrambled or luciferase siRNA, and with 50 nM or 100 nM of Chk-α- or PD-L1-specific siRNA for individual treatments. For combination siRNA treatments, 50 nM of each specific siRNA was used. Cells were treated with siRNA for 48 h because this incubation period resulted in the most effective downregulation of the target genes. D-FECT 4 (Dharmacon, Catalog Item T-2004-03) was used as the transfection agent for MDA-MB-231, Pa09C and Pa20C cells, and Lipofectamine 2000 (Thermo Fisher, Waltham, MA, USA, Catalog Item 11668019) for SUM-149 cells. All transfections were carried out based on established protocols [34 (link)].
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