PARP1 stable knockdowns and corresponding controls were generated using Amaxa® Nucleofector® Technology in the Laboratory of Transcriptional Regulation, Institute for Medical Biology, PAS, Lodz, and with the kind help of prof. Łukasz Pułaski. In brief, 0.5 µg of PARP1 shRNA Plasmid and Control shRNA Plasmid-A were mixed with THP1 cells suspended in Nucleofector Solution, subjected to electroporation with Amaxa® Nucleofector® according to the manufacturer’s instructions, and immediately diluted with warm RPMI with 10% FBS. After 24 h in culture, cells were selected with puromycin (5 µg/mL) for a month. After selection, puromycin was added to cells every second week.
The transient PARP1 overexpression was carried out as described previously [53 (link)]. In brief, THP1 cells at a density of 1,000,000/mL were treated with the complexes of pCMV3-EMPTY or pCMV3-PARP1 vectors and transfection reagent ViaFect. After 24 h, cells were cultured for 24 h as described in 2.2 and then subjected to LPS treatment (±olaparib).
The transient PARP1 overexpression was carried out as described previously [53 (link)]. In brief, THP1 cells at a density of 1,000,000/mL were treated with the complexes of pCMV3-EMPTY or pCMV3-PARP1 vectors and transfection reagent ViaFect. After 24 h, cells were cultured for 24 h as described in 2.2 and then subjected to LPS treatment (±olaparib).
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