Cultivation and siRNA treatment of THP1 cells (DSMZ GmbH, Braunschweig, Germany) was performed as previously reported [7 (link)]. Briefly, the cell line was authenticated via MLL-AF9 breakpoint sequencing, was routinely checked for mycoplasma contamination and transfected with 50 nM Silencer Select siRNAs (Life Technologies, Carlsbad, CA, USA) and Dreamfect (OZ Biosciences, Marseille, France) leading to transfection efficiencies and survival rates of 93 % each. Experimental incubations lasted eight days with repeated transfections on day 0, 3, and 6. Prior to each transfection event, cell densities were determined and cells were reseeded at 5 × 104 cells per ml [7 (link)].
THP1 miRNA mimic transfections were performed as described for siRNA transfections but with 30 nM Ambion Pre-miR miRNA Precursors (hsa-miR-511-5p AM10237, neg Control #1 AM17110, neg Control #2 AM17111) and experimental incubations up to day nine again with repeated transfections on day 0, 3, and 6.
Human CD14+ monocytes (C-12909, PromoCell GmbH, Heidelberg, Germany) were cultivated for 48 h in mononuclear cell medium (PromoCell) prior to immunostaining and RNA isolation. Human CD34+ progenitor cells (C-12921, PromoCell) were directly taken to RNA isolation.
THP1 miRNA mimic transfections were performed as described for siRNA transfections but with 30 nM Ambion Pre-miR miRNA Precursors (hsa-miR-511-5p AM10237, neg Control #1 AM17110, neg Control #2 AM17111) and experimental incubations up to day nine again with repeated transfections on day 0, 3, and 6.
Human CD14+ monocytes (C-12909, PromoCell GmbH, Heidelberg, Germany) were cultivated for 48 h in mononuclear cell medium (PromoCell) prior to immunostaining and RNA isolation. Human CD34+ progenitor cells (C-12921, PromoCell) were directly taken to RNA isolation.
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