Allstars control sirna

AGS-EBV cells were plated in a 6-well dish at 20% confluency and transfected with either 40 pmoles of siSUMO1 (Santa Cruz) or Allstars control siRNA (Qiagen) using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. The transfection was repeated after 24 and 48 hours. 24 hours later, cells were lysed in 9M urea buffer for Western blot analysis or fixed and stained with anti-BZLF1 for fluorescence microscopy analysis. Lysates were sonicated and clarified by centrifugation. 80 μg of clarified lysate were analyzed by Western blotting with SUMO1, BZLF1 and actin antibodies. The percentage of reactivated cells was determined by performing immunofluorescence microscopy for BZLF1 (as described below), and counting ~300 cells for each sample in three independent experiments.

hESCs maintained on Matrigel at 5% oxygen were passaged and incubated overnight. For each transfection, 50 nM siRNA (CTBP1/2 [Ambion]; CTBP1 [Ambion]; CTBP2 [Ambion]; HIF-2α [QIAGEN]), along with 12 μL INTERFERin for CTBP siRNAs (Polyplus) or HiPerFect for HIF-2α siRNA (QIAGEN) transfection reagent were mixed in 200 μL of KnockOut DMEM (Invitrogen) and added in a drop-wise manner to 1 well of a 6-well plate. Cells were harvested 48 h post-transfection and RNA or protein extracted. Allstars control siRNA (QIAGEN) that has no homology to any known mammalian gene was used as a negative control for each transfection.
For double knockdowns (CTBP1+2 siRNA), 50 nM of each siRNA and 12 μL InterferIN transfection reagent were added to 200 μL of KnockOut DMEM. Twice the volume of Allstars negative control siRNA was added to the controls.

DLD1, SW480 and HT29 cells were seeded at 2x10⁴ cells/cm², cultivated on 4 well-chamber slides (Sigma C7057, Taufkirchen, Germany) over-night and treated with anti-EGFR monoclonal antibody cetuximab-protamine or cetuximab alone incubated with Alexa Fluor 488-labeled Allstars control siRNA (Qiagen 1027284, Hilden, Germany) and Allstars negative control siRNA-Alexa 555 (Qiagen 1027286, Hilden, Germany) at 1:10 molar ratio each over night at 37 °C and 5% CO₂. Subsequently, cells were washed with phosphate buffered saline (PBS), fixed with ice-cold paraformaldehyde (PFA) 4%, stained with Hoechst, mounted with Dako fluorescent mounting medium and photographed on a Zeiss Axioskop.