On targetplus human sirna

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.

The synthetic miRVana^TM hsa-miR-15a-5p mimic (#MC10235) and hsa-miR-15a-5p inhibitor oligonucleotides (#MH10235) were purchased from Life technologies. A total of five million K562 or KG1a cells were nucleoporated using Amaxa^® Nucleofector^® Technology (Lonza) with 100 µl of solution V or solution L, respectively (program T-016 for K562 and V-001 for KG1a) and with 750 pmol of precursor oligonucleotide and cultured for 24 h or 48 h. Scrambled oligonucleotide, miRVana^TM miRNA Mimic Negative Control #1 (#4464058), was used as control. For siRNA transfection, a total of five million K562 cells were nucleoporated using the same method with a combination of four ON-TARGETplus human siRNAs against four target genes (ATG9A: #L-014294-01, ATG14: #L-020438-01, GABARAPL1: #L-014715-00, and SMPD1: #L-006676-00, Dharmacon, Lafayette, CO, USA) at a concentration of 25 nM for each siRNA or with 100 nM of ON-TARGETplus Non-targeting pool (#D-001810-10, Dharmacon, Lafayette, CO, USA) as control.

MDA-MB-231 and Hs578t cells were seeded on 6 well plates, incubated, and allowed to attach overnight. After 24 h, cells were transfected using 50 nM ON-TARGET plus Human siRNAs from Dharmacon for either Nek2 (L-004090-00-0005), Slug/SNAI2 (L-017386-00-0005), Zeb1 (L-006564-01-0005), or silencer negative control siRNA (Invitrogen AM4611) and mixed with jetPRIME Transfection Reagent (Polyplus 114-07) in cell media according to manufacturer instructions. Transfection complex was allowed for 48 h and used for the experiments described below.