Interferin transfection reagent

The plasmids for p300-siRNA or control-siRNA (40 nM; Bioneer, Daejeon, Korea) were transiently transfected into MCF-7 cells for 24 h, using an INTERFERinTM transfection reagent (Polyplus- transfection Inc., New York, NY), according to manufacturer’s protocols.

Scrambled controls and p62 and IRE1 small interfering RNA (siRNA) were obtained from Invitrogen (169688). IRE1 siRNAs were obtained from Bioneer. Transfection with these siRNA plasmids was performed using the Interferin^TM transfection reagent (Polyplus-transfection Inc., 11062400) according to the protocol provided by the manufacturer.

A549/CR and H460/CR cells were transfected with control or p53 siRNAs using Interferin^TM transfection reagent (Polyplus-transfection Inc., New York, NY, USA). Luciferase activity was determined using a p53 luciferase reporter assay kit and a LUMIstar OPTIMA luminometer plate reader (BMG Labtech, Offenburg, Germany) after A549/CR and H460/CR cells were transiently transfected with p53 reporter gene and exposed to PGG (12.5 and 25 μM) for 48 h.

Cells were transiently transfected with a validated scrambled control small interfering (si)RNA, or siRNA specifically for L11 or c-Myc (Santa Cruz Biotechnology, Santa Cruz, CA) or Flag–tagged L11 overexpression vector kindly provided by Prof. Hua Lu (School of Medicine, Oregon Health and Science University, USA) by using Interferin^TM transfection reagent (Polyplus-transfection Inc., New York, NY). Briefly, the mixture of siRNA or Flag–tagged L11 overexpression vector with Interferin^TM transfection reagent was incubated for 10 min, added to each well of the cells (siRNA final concentration = 40 nM) and incubated at 37 °C for 48 h before drug treatment.

Cells (1 × 10⁵) were seeded on a 6-well plate and transfected with 15 nM of control or Sp1 siRNA (Santa Cruz Biotechnology, Inc., Danvers, MA, USA) for 48 h using INTERFERin transfection reagent (Polyplus-transfection Inc., New York, NY, USA) according to the manufacturer’s protocol.

All siRNAs were purchased from Dharmacon as ON-Target plus. THP-1 cells were transfected by electroporation with indicated gene-specific siRNAs or non-specific siRNAs using Neon (Invitrogen). Lysates were collected 72 h after transfection for western blotting and qRT-PCR analysis. For nucleic acid stimulation, PMA-differentiated THP-1 cells were transfected with 5 μg/ml of poly (I:C), poly (dA:dT), or isolated DNA or RNA, using Lipofectamine 2000 transfection reagent (Invitrogen) and undifferentiated THP-1 cells were transfected by electroporation with 10 μg/ml of isolated RNA using Neon. Cells were harvested 4 h post transfection, followed by RNA isolation. For reconstitution of SAMHD1-deficient THP-1, THP-1 cells were transfected by electroporation with expression vectors for wild-type or mutant SAMHD1 using Neon. Isolated PBMCs were transfected using Interferin™ transfection reagent (Polyplus-transfection Inc.), according to the manufacturer’s instructions.

The human TCL1-expressing B-cell lines 697, RAJI and EHEB were maintained in RPMI 1640 medium, supplemented with 10% fetal bovine serum. Mouse splenocytes were freshly isolated from diseased mice, using the following procedure: spleens were explanted from euthanized mice and placed in cold phosphate-buffered saline (PBS) solution. Cell suspension was obtained by squeezing spleen between two slides and washing with PBS. Erythrocytes were degraded by using ammonium chloride (0.8%) with EDTA (0.1 mM) (Sigma). White cells were washed, passed through a 70-μm filter and counted. Depending on the experimental procedure, the B-cell pool was further purified by using B220-conjugated magnetic beads (Invitrogen) following the manufacturer's instructions. Leukemic splenocytes were cultured in 48 multi-well plates, using RPMI 1640 medium supplemented with 10% fetal bovine serum, 10 mM HEPES buffer and 1 mM Na pyruvate. Transient transfections were performed with 100 nM pre-miR (Ambion) or single-stranded mimic (IDT) or 20 nM anti-TCL1 siRNA (Dharmacon), or scrambled negative controls, diluted in Opti-MEM serum-free medium (Gibco) and complexed with INTERFERin transfection reagent (Polyplus), following the manufacturer's instructions. Cells were harvested 72 h after transfection and were either lysed in RIPA buffer for Western blot analysis or stained for FACS analysis.