On targetplus sirna

Approximately 500,000 cells were transfected using a final concentration of 20 nM EPSTI1 (J-015094-09-0020 and J-015094-12-0020) or non-targeting (D-001810-01-20 and D-001810-02-20) ON-TARGETplus siRNAs from GE Healthcare Dharmacon using 20 µL of Lipofectamine RNAiMAX (ThermoFisher #13778–150) and 500 µL OptiMEM (ThermoFisher #31985070). Cells were incubated for 72 hr before further analysis.

Colorectal cancer cell line HCT116 and isogenic variants (MUS81 null and EME1 haplo-insufficient)^{25 (link)} were provided by Prof. Kiyoshi Miyagawa (University of Tokyo, Japan). Esophageal cell lines OE33 and Flo1 were obtained from ECACC. For siRNA depletion, cells were treated 96 and 48 hours prior to plating using ON-TARGETplus siRNA (GE Lifesciences, Dharmacon, USA) to MUS81 and XPF, and Lipofectamine® RNAiMAX transfection reagent (Thermofisher), following manufacturers’ instructions (full details in Supplementary Information).

CAD mouse neuroblastoma cells (42 (link)) were cultured in Dulbecco's Modified Eagle Medium/High Glucose (DMEM; Thermo Fisher Scientific/GIBCO), supplemented with 11% FetalClone III Serum (GE Healthcare/HyClone), 1 mM sodium pyruvate (Thermo Fisher Scientific/GIBCO), 100 IU/ml penicillin and 100 μg/ml streptomycin, at 37°C in the presence of 5% CO₂. For transfection experiments, cells were plated in the CAD medium without antibiotics at a density of 4 × 10⁵ cells per well of a six-well plate for tissue culture. Twelve hours post-plating, cells were transfected with corresponding ON-TARGETplus siRNA (GE Healthcare/Dharmacon; siControl [D-001810-01-20]; siPtbp1 [J-042865-11-0050]; siPtbp2 [L-049626-01-0005]) using Lipofectamine RNAiMAX (Thermo Fisher Scientific/Invitrogen). Following 36-hour incubation, cell cultures were typically re-transfected with 1 μg of a minigene plasmid using Lipofectamine 2000 and incubated for another 36 h prior to RNA harvest. For U1 snRNA suppressor experiments, the ratio of pN/S6U1 plasmid to minigene plasmid used for transfections is 9:1. Neurons, neuronal progenitor cells and astrocytes were isolated from mouse cortices and maintained as described elsewhere (43 (link)).

Small interfering RNA (siRNA) was carried out as previously described with slight modifications7 (link)56 (link). AhR siRNA was the SMARTpool: ON-TARGETplus siRNA from GE Healthcare, UK. The control siRNA was the ON-TARGETplus Non-targeting control from GE Healthcare. Monocytes were transfected on day 0 with either the specific AhR siRNA or a control scrambled (non-specific siRNA) (50 nM) using the HiPerFect Transfection Reagent. The inhibition of target gene was assessed at day 6.

A sub-library of ON-TARGETplus siRNA (SMARTpools; GE Healthcare) was individually transfected into WT Vero cells as described [26] (link). Each transfection experiment included a non-targeting control (NTC) siRNA, a siRNA targeting RV3 (positive control), and siTOX (GE Healthcare) served as a transfection control where transfection resulted in cell death. All siRNAs were transfected to a final concentration of 50 nM.