Sirna targeting

Dasatinib and Erlotinib were purchased from LC Laboratories (Woburn, MA, USA), 17-AAG and LY294002 were purchased from EMD Chemicals (Gibbstown, NJ, USA), respectively. Unless otherwise indicated, chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). The constitutively active Src mutant (Src (Y527F)) expression vector was kindly provided by Dr. Faye M. Johnson (MD Anderson Cancer Center, Houston, TX, USA). The HSP90 expression vector was kindly provided by Dr. William C. Sessa (Yale University School of Medicine, CT, USA). siRNA targeting Src was purchased from Dharmacon.

U251-derived TMZ-resistant cells were plated at 10⁵/mL in 6-well plates in DMEM media without antibiotics. Twenty-four hours later, the cells were transfected with an optimized amount (5 nmol/L) of siRNA targeting human RAD51 (Dharmacon, Lafayette, CO, USA) or non-targeting siRNAs using DharmaFECT reagent (Dharmacon) according to the manufacturer’s protocol as described previously [13 (link)].

The knockdown of G9a or GLP individually or simultaneously using siRNA was performed in 6-well or 12-well plates as previously described [28 (link)]. Briefly, siRNA targeting G9a (Cat #: L-053728-01-0005) or GLP (Cat #: L-059041-01-0005) was purchased from Dharmacon (Lafayette, CO, USA). Media changes were performed every 24 h, and nontargeting siRNA acted as a negative control for the knockdown experiments. Transfection with 50 nM siRNA was repeated after the initial 24 h incubation. Cells were harvested 72 h following the initial transfection. Extracts for protein analyses were prepared by collecting cells in immunoprecipitation (IP) buffer (10 mM Tris (pH 7.4), 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% IGEPAL CA-630, protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µM pepstatin, 50 trypsin inhibitory milliunits of aprotinin, 10 µM leupeptin, 1 mM 1,10-phenanthroline), and phosphatase inhibitor (0.2 mM sodium vanadate)). To extract RNA for gene expression analyses, cells were harvested in buffer provided in the RNeasy mini kit (Qiagen, Hilden, Germany). As a positive control to assess siRNA transfection efficiency, a knockdown of Cyclophilin B (Dharmacon, Lafayette, CO, USA; Cat #: D-001820-02-05) was also performed [28 (link)].

CLL cells were transfected using the Amaxa nucleofection technology (Amaxa) as previously described^{10 (link)} with 3 µg siRNA targeting Bcl-XL and non-targeting siRNA (Ambion, Thermo Fisher Scientific) or 3 µg siRNA targeting NIK and non-targeting siRNA (Dharmacon). After nucleofection, cells were cultured on 3T40L for 24 h before drugs sensitivity assay was performed or protein lysates were obtained.