Lipofectamine rnaimax

In most experiments cells were seeded 1 day before being transiently forward transfected with RNAi (20 nM) directed against either APP, Lf, ARF6, DYM, Rab5a, Rab4a, Rab7a, Rab11a (SMARTpool: ON-TARGETplus Human siRNA; Horizon Discovery), or control non-target (ON-TARGETplus Non-targeting Pool; Horizon Discovery) using 4 μl of Lipofectamine^® RNAiMAX (Life Technologies) as described in the manufacturer’s instructions. In the case of HMC3, cells were re-plated into transwell inserts prior to activation by IFN-γ treatment and the inserts were subsequently placed into wells containing adhered wt-APP⁶⁹⁵ SH-SY5Ys.
Dual depletion of Rab4a and Rab11a required reverse transfection with Rab4a (20 nM; SMARTpool: ON-TARGETplus Human siRNA; Horizon Discovery) followed by forward transfection with Rab11a (20 nM; SMARTpool: ON-TARGETplus Human siRNA; Horizon Discovery) using 4 μL of Lipofectamine^® RNAiMAX for each treatment. Cells were incubated with the RNAi mixture for 48 h.
Lastly, a reverse transfection procedure was required for RNAi directed against CHC (40 nM; SMARTpool: ON-TARGETplus Human siRNA; Horizon Discovery) using 9 μL of Lipofectamine^® RNAiMAX (6 h at 37 °C). Media was replaced with complete growth medium and cells were incubated for a further 72 h.

For PHC2 and PHC3 knockdown 100,000 HeLa cells were reverse transfected with either 50 nM of PHC2 and PHC3 siRNAs individually (Dharmacon) or in combination for 96 h using Lipofectamine RNAiMax as per manufacturer instructions. Equal amounts of whole cell lysate were separated on 10% SDS–PAGE gels. Blots were probed with primary antibodies (H2Aub(K119), Cell Signaling Technology; H3 (Abcam); β-actin, EMD Millipore). PHC2 and PHC3 siRNA mediated knockdown efficiency was measured by quantitative real time PCR. Uncropped images of the western blots are shown in Supplementary Fig. 14.

Approximately 1 × 10⁶ A549 cells were transfected with 20 nM ON-TARGETplus Non-Targeting Control Pool (Dharmacon, D-001810-10-20) or 20 nM siGENOME Human ANP32A siRNA SMARTpool (Dharmacon, M-016060-00-0005) and siGENOME Human ANP32B siRNA SMARTpool (Dharmacon, M-020148-01-0005) using Lipofectamine RNAiMax and Opti-MEM according to the manufacturer’s instructions. Forty-eight hours posttransfection, cells were either lysed for Western blot analysis or infected with influenza A virus/WSN/33 (H1N1) virus.
For infections, cells were washed with PBS prior to infection, followed by infection with influenza A/WSN/33 (H1N1) virus at an MOI of 1 in DMEM supplemented with 0.3% BSA for 15 h. Total RNA was extracted using TRIzol (Invitrogen) and reconstituted in 20 μl of nuclease-free water.