On targetplus non targeting pool sirna

To silence gene expression, glioma cells were transiently transfected with siRNA molecules targeting specifically HLA-E [14 (link)]: (535–553): 5’-GCCUACCUGGAAGACACAU(dTdT)-3’ and 5’-AUGUGUCUUCCA-GGUAGGC(dTdT)-3’, for nectin-2 gene silencing ON-TARGETplus nectin-2 siRNA (Dharmacon, Lafayette, CO) was applied. ON-TARGETplus Non-Targeting Pool siRNA (Dharmacon) was used as a control in all gene silencing experiments. Glioma cells were transfected with 2 nmol siRNA through electroporation with one pulse (1500 mV, 10 ms), using a Neon^® Transfection system (Invitrogen). The result of gene silencing was verified by flow cytometry at 24 h after transfection.

To better assess the role of the PIM kinases in MM cell lines, short hairpin RNA and small interfering RNA (siRNA) was used to knockdown each of the kinases in U266, MM1.S, RPMI and Dox40 cells. Transient transfection-mediated siRNA knockdown was carried out in MM1.S and U266 cells using the Amaxa Cell Nucleofector Kit (Lonza, Basel, Switzerland). On-Target Plus human siRNA for PIM1, PIM2 and PIM3, and On-Target Plus Non-Targeting Pool siRNA was used as a scramble control (Dharmacon, Lafayette, CO, USA). Inducible PIM2 short hairpin RNA knockdowns were cloned in pLKO-Tet-ON plasmids (Addgene, Cambridge, MA, USA) with the following PIM2 primers: F, 5′-CCGGCCAGGATCTCTTTGACTATATCTCGAGATATAGTCAAAGAGATCCTGGTTTTT-3′ and R, 5′-AATTAAAAACCAGGATCTCTTTGACTATATCTCGAGATATAGTCAAAGAGATCCTGG-3′ (Life Technologies). PIM2 short hairpin RNA or pLKO.1 control plasmids were co-transfected with pVSV-G and delta 8.9 plasmid into 293 T cells with FUGENE 6 transfection reagent (Roche, Indianapolis, IN, USA). Virus was then collected at 48/72 h and applied to MM cell lines via spinoculation. Knockdowns were induced with administration of doxycycline at a dose of 100 ng/ml (Selleck Chemicals).