C sirna

All cells used in this study were purchased from ATCC, if not stated otherwise. Human lung adenocarcinoma cell lines H522 and H1650 were propagated in RPMI medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco). Human osteosarcoma cell line U2-OS and its derivatives were maintained in DMEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco). Cells were grown at 37°C in a humidified atmosphere with 5% CO₂. U2-OS cells with Tet-inducible expression of shRNA against Set7/9 and the reference cell line were generated as described in [45 (link)]. To manipulate with the expression level of Mdm2 specific siRNA (siRNA-Mdm2) as well as the control one (c-siRNA) were purchased from Ambion Life Technology (Cat#4390824, ID S8630). Typically, 50nM of c-siRNA or siRNA-MDM2 were used for transfection with Lipofectamine 2000 (Life technologies, USA) in Opti-MEM (Gibco, USA). Following two days of incubation to achieve knockdown of Mdm2 expression, cells were treated with doxorubicin as described.

TM9SF4 gene was silenced with synthesized small interfering ribonucleic acids (siRNA) TM9SF4-siRNA (Sil. Sel. siRNA TM9SF4 human, INV, STD, 5NM, cod. 4392420 from Life Technologies, Italy). 1,5x10⁶ U937 cells were transfected with 100 nM of TM9SF4-siRNA, or non-targeting control siRNA (c-siRNA) (Sil. Sel. siRNA Negative Control, 1.5 NM, cod. 4390843, from Life Technologies, Italy) by using Lipofectamine 3000 according to the manufacturer’s protocol (Life Technologies, Italy). U937(TM9SF4-siRNA)- and U937(c-siRNA)- transfected cells were maintained 48 hrs in culture under normoxia; TM9SF4 downmodulation was controlled at protein level by Western blot analysis.
HIF-1α gene was silenced with synthesized HIF-1α-siRNA (ON-TARGET plus SMART pool siR J004018-10 HIF-1α, from Dharmacon, CO, USA). U937 cells were transfected twice with 100 nM of HIF-1α-siRNA, or non-targeting control siRNA (c-siRNA) (ON-TARGET plus Non-Targeting Pool D-001810-10-05, from Dharmacon, USA) by using Lipofectamine 3000. Following the second transfection, U937(HIF-1α-siRNA)- and U937(c-siRNA)- transfected cells were maintained 24 hrs in culture under hypoxia (1% O₂), as described [46 (link)]. HIF-1α downmodulation was controlled by Western blot analysis of nuclear extracts prepared from U937(HIF-1α-siRNA) cells, as compared to U937(c-siRNA) control cells.

Gene silencing was performed in PNTs and H727 cells using species-specific human FLNA pre-designed siRNA (#4392420, Invitrogen) and Rap1 siRNA (#sc-36384 Santa Cruz), using Lipofectamine 2000 transfection reagent (#11668-019, Invitrogen) according to the manufacturer's instruction, for 72h and 48h of incubation, respectively.
In order to obtain the best efficiency of FLNA silencing three different human FLNA silencer select pre-designed siRNAs were tested.
Preliminary experiments to determine the optimal concentration of siRNAs and the kinetics of silencing of FLNA and Rap1 were performed. A negative control siRNA (C- siRNA) (AM4611, Invitrogen), a non-targeting sequence without significant homology to the sequence of human, mouse or rat transcripts, was used in each experiment. Western blotting was performed in each experiment to control the expression level of FLNA and Rap1 in silenced cells.