Sirna oligonucleotides

For isolation of mouse macrophages, mice received an intraperitoneal injection of thioglycollate (4%). Cells were collected after 4 days by peritoneal lavage, centrifuged, resuspended in RPMI medium (10% FBS) and plated onto μ-dishes (Ibidi) for 24 h.
Human THP-1 monocytes were stimulated with PMA (300 nM, Merck) overnight to differentiate them into macrophages, which were then transfected with SR-BI-specific siRNA oligonucleotides or control oligonucleotides (Qiagen) for 48–72 h. Human neutrophils were isolated from whole blood using a density gradient separation method with dextran (3%, MW 500,000, Roth) containing HBSS/Hepes (10 mM) medium. After centrifugation, separation of the neutrophil layer and lysis of residual erythrocytes, the neutrophils were washed and resuspended in RPMI/Hepes (10 mM). Neutrophil apoptosis was induced by overnight incubation with pyocyanin (25 μM, Sigma). The apoptotic neutrophils were stained with TAMRA (50 μM, Thermo Fisher Scientific) and incubated for 30 min with macrophages at 37 °C.
CHO cells were stably transfected with the cDNA for human SR-BI14 (link). Transient transfections with different plasmids for murine SR-BI (native mSR-BI and its mutants SRCDSR and CDSRCD27 (link), donated by Margery Connelly), were performed using Lipofectamine^®2000 (Thermo Fisher Scientific).

siRNA oligonucleotides targeted specifically to rat DDAH 1 and NOX-4 (Qiagen, Hilden, Germany) were used in this experiment. Cells were seeded on 6-well plate in serum free media for overnight. HiPerfect transfection reagent (Qiagen, Hilden, Germany) were used to transfect the cells with 20 nM of respective siRNAs as per the manufacturer’s instructions. Scrambled siRNA (20 nM) were used as transfection control. After the respective treatments, cells were harvested using RIPA cell lysis buffer for studying protein expressions.

RNAi knockdown of OLMALINC was carried out using four commercially prepared siRNA oligonucleotides (Qiagen) specific to different fragments of the OLMALINC transcript. Briefly, MO3.13 and SK-N-SH cells were seeded at 40% confluence in 6-well plates in antibiotic-free media and equimolar mix of four oligonucleotides was transfected using Lipofectamine 2000 and Opti-MEM I (Invitrogen) following the manufacturer’s protocol. For validation experiments MO3.13 oligodendrocytes were transfected with two individual siRNAs used previously in the four siRNA mix. Transfection efficiency was confirmed by substituting the siRNA with fluorescently labelled BLOCK-iT reagent (Invitrogen), using the same transfection procedure and this confirmed >95% transfection efficiency was achieved as assessed by fluorescence microscopy.

All small interfering RNA (siRNA) oligonucleotides in a purified and annealed duplex form were purchased from Qiagen (Valencia, CA 91355). The targeting sequences are as follows: UBXD7 (SI00455364, catalog number), 5′-CAGCACGTGCATATTCATTTA-3′; UFD1 (SI04132583), 5′-CACTGGATGATGCAGAACTTA-3′ VCP/p97 (SI030197300), 5′-AACAGCCAUUCUCAAACAGAA-3′ and control (Ctrl) siRNA (SI03650325), 5′-AAUUCUCCGAACGUGUCACGU-3′.