On targetplus smartpool small interfering rna sirna

To knock down IDO, the ON-TARGETplus SMART-pool small interfering RNA (siRNA) (L-010337-01-0005; GE Dharmacon, Lafayette, CO, USA) was used. The sequences were as follows: Human IDO1, NM_002164, sense, 5′-UCACCAAAUCCACGAUCAUUU-3′, antisense, 5′-PUAUGCGAAGAACACUGAAAUU-3′; sense, 5′-UUUCAGUGUUCUUCGCAUAUU-3′, antisense, 5′-PUAUGCGAAGAACACUGAAAUU-3′; sense, 5′-GUAUGAAGGGUUCUGGGAAUU-3′, antisense, 5′-PUUCCCAGAACCCUUCAUACUU-3′; and sense, 5′-GAACGGGACACUUUGCUAAUU-3′, antisense, 5′-PUUAGCAAAGUGUCCCGUUCUU-3′. The ON-TARGETplus siCONTROL Nontargeting Pool (D-001810-10-05; GE Dharmacon) was used as the negative control. siRNAs were transfected into MSCs by the Amaxa Nucleofector II device (Lonza) and the Human MSC Nucleofector kit (Lonza). Briefly, aliquots of 5 × 10⁵ MSCs were re-suspended in 100 μl of the Nucleofector solution in the presence of 1.5 μg of IDO or control siRNA and then electroporated by the U-23 program. Cells were immediately transferred into 37 °C pre-warmed complete culture medium and plated onto six-well dishes. After 72 h from transfection, cells were harvested and lysed for the immunoblotting measurement of IDO levels, while the supernatants were recovered for the assessment of IDO activity and to be used in functional assays.

For RNAi assays, H1R or H2R On-Target plus Smart pool small interfering RNA (siRNA; Dharmacon) or negative control siRNA (50 nM) were delivered using the DharmaFECT transfection reagent as recommended by the manufacturer. At 48 h post-transfection, the cells were induced by Dox (0.5 μg/mL) in combination with histamine (100 μM) for additional 48 h, then protein expression was measured by Western blot.