On targetplus control pool

Leukemic cell lines were transfected with the Neon transfection system (Life Technologies, Carlsbad, CA, USA). Cells (2 × 10⁶) were re-suspended in 100 μl of resuspension buffer, and various doses of RNA were added. RNA sequences were: specific STAT5A and STAT5B siRNA (ON-TARGETplus SMARTpool, human STAT5A and STAT5B, Dharmacon, Lafayette, CO, USA) or siRNA control (sigenome control pool non targeting #2, or ON-TARGETplus control pool, Dharmacon, Lafayette, CO, USA), anti-miR control or anti-miR-16 (Life Technologies, Carlsbad, CA, USA), and pre-microRNA negative control or pre-microRNA-16 (Life Technologies, Carlsbad, CA, USA). Cells were then transfected with the nucleofector device (program 5 for MOLM14, MV4-11 and OCI-AML3 cell lines, custom program for the HEL cell line), and subsequently re-suspended in normal culture medium at a concentration of 1 × 10⁶ cells/ml.

THP-1 cells (10⁵ cells/ml) were differentiated into macrophage like cells with PMA (50 ng/ml), as mentioned earlier. The PMA treated cells were then transiently transfected with 600 pmole [26 (link)] of siGENOME Human HDAC1 (3065) siRNA–SMARTpool (Dharmacon, USA). Lipofectamine 3000 (Invitrogen, USA) was used to transfect the siRNA as per manufacturer’s instructions. Briefly, the transfection reagents were mixed in RPMI media without FBS. The cells were incubated with siRNA for 24 h to allow gene silencing. Subsequently, the cells were washed and incubated with L. donovani at 20:1 MOI. The cells were harvested 6 h post-infection. Total RNA was extracted, followed by cDNA synthesis. The mRNA expression levels of HDAC1 and the defense genes were analyzed by qRT-PCR, as mentioned above. ON-TARGET plus Control Pool (Dharmacon, USA) was used as a negative control. The transfection efficiency was 75–80% as has also been reported by the manufacturer (Dharmacon, USA).

At 50% confluency, NRK cells were transiently transfected with 50 nM OMA1 (siGENOME SMARTpool, Dharmacon; target sequence: GUAGGACUCUCAAGAACAA; UUGAAUAGCCUUCGUGCUU; GACAUACGCACUUGGAAA; GGCAAUGCCUUCGUGCUU) using siRNA transfection reagent (Invitrogen, Waltham, MA, USA) in OPTI-MEM (Invitrogen) with Lipofectamine RNAiMAX and Lipofectamine 2000 for 24 and/or 48 h at 37 °C as previously described [34 (link)]. The same concentration of scrambled siRNA (ON-TARGET plus Control Pool, non-targeting pool, Dharmacon; target sequences: UGGUUUACAUGUCGACUAA, UGGUUUACAUGUUGUGUGA, UGGUUUACAUGUUUUCUGA, UGGUUUACAUGUUUUCCUA) was used as a control. The next day, CS + RW or control treatments were assigned. Efficiency of OMA1 transfection and knockdown was assessed by western blotting as previously described [34 (link)].

3T3-L1 preadipocytes were seeded in 12-well plates (10⁵ cells per well) and transfected with a pool of nontargeting siRNA control oligonucleotides (ON-TARGET plus Control pool, D-001810-10-05; Dharmacon), or siRNA oligonucleotides against mouse MOR23 (80 nM, ON-TARGET plus smart pool, L-022371-02; Dharmacon, Lafayette, CO, USA) using Lipofectamine 2000 (Life Technologies, Carlsbad, CA, USA). Target sequences for siRNA against mouse MOR23 were as follows: CAAUGGGUUAUGAUCGUUA, CUGAAGUGAUAGAGUUCGU, GGUGUAAGUUCAUUUGUAA, and CCAUAGGGCUGAUAUUUAU. Forty-eight hours after the transfection, the cells were stimulated for adipogenesis. The MOR23 ON-TARGET plus smart pool was a mixture of four siRNAs. The knockdown efficiency of siRNA was assessed by semiquantitative RT-PCR.

The PMA treated differentiated THP-1 cells (10⁵ cells/ml) were transiently transfected with 600 pmole (Garcia-Garcia et al., 2009 (link)) of siGENOME Human HDAC1 (3065) siRNA – SMARTpool (Dharmacon, USA) using lipofectamine 3000. The cells were incubated with siRNA for 24 h to allow gene silencing. Subsequently, the cells were washed for infection and stimulated as described earlier. THP-1 cells were harvested after 6 h and mRNA expression of HDAC1 and CIITA genes was analyzed by qPCR. ON-TARGET plus Control Pool (Dharmacon, USA) was used as a negative control. The basal level of HDAC1 and CIITA in uninfected Sc-siRNA transfected cells respectively were used for data normalization and were taken as 1.0. The transfection efficiency was calculated (ThermoFisher) to be >50%, as has also been reported by the manufacturer (Dharmacon, USA).

1.5×10⁵ cells per well were seeded in serum and antibiotic free RPMI medium (24-well format). Cells were transfected according to Lipofectamine RNAiMAX reagent protocol (2013), using 10 pmol siRNA human ADAM17 (pool of three stealth RNAi: HSS110434, HSS110435, HSS186181; invitrogen/life technologies/Thermo scientific) or Control siRNA (ON-TARGETplus Control Pool, Dharmacon) and Lipofectamine® RNAiMAX transfection Reagent (Invitrogen, #13778) in two sequential transfection steps at day one and day two after seeding. 6 hours after the second transfection cells were incubated with 6 μM cisplatin or NaCl. Following 24 h at 37 °C supernatants were stored and cells harvested as described above. Supernatants were tested on AREG-levels, see below.