Smartpool sirna oligonucleotides

SMARTpool siRNA oligonucleotides were purchased from Dharmacon (Lafayette). Transfection was carried out as described in [19 (link)]. Twenty-four hours after transfection, fresh medium was added. At 80 h cells were either left untreated or treated with 40 ng/ml nocodazole for 16 h, then harvested by mitotic shake-off and analysed as described in the figure legends. Alternatively, 24 h after transfection, cells were irradiated with 10 Gy and harvested 1 h post-recovery as described above.

Lamin A/C depletion in A549 cells and CHMP7 depletion in HT-1080 cells was accomplished using DharmaFECT (Dharmacon) and target-specific siRNA according to the manufacturer’s protocol, with final siRNA concentrations of 2.5 nM (LMNA) and 100 nM (CHMP7). SmartPool siRNA oligonucleotides, containing four target sequences in one mix to reduce off-target effects, were purchased from Dharmacon (GE Healthcare): human LMNA (ON-TARGET plus SMART pool, L-004978-00), human CHMP7 (ON-TARGETplus SMARTpool, L-015514-01), and non-targeting control siRNA (ON-TARGETplus non-targeting pool, D-001810-10).

Human dermal microvascular endothelial cells (HMVECs) and human umbilical vein endothelial cells (HUVECs) were from Lonza (Walkersville, MD). Cells were cultured in endothelial growth medium supplemented according to the manufacturer’s instructions. MyEnd cells (15 (link)) were cultured, as previously described (6 (link)). HMVECs were depleted of Epac1 or Mena/VASP with SMARTpool siRNA oligonucleotides from Dharmacon (Lafayette, CO). Briefly, 100 nmol/L siRNA mix were complexed with OligofectAMINE reagent (Thermo Fisher Scientific, Parsippany, NJ) and cells were exposed to the lipid-oligonucleotide mixture for 4–6 h. Transfection efficiency was evaluated by Western blotting.

Small interfering RNA (siRNA) mediated gene knockdown was performed using Hyperfect transfection reagent (Qiagen, Hilden, Germany) following the manufacturer's instructions. A total of 20 pM siRNA were transfected per 2 × 10⁴ cells. Cells were grown for 72 h before IR. SMARTpool siRNA oligonucleotides from Dharmacon were used for Control, HP1α, HP1β, HP1γ, CtIP, EXO1 and BLM. SUV39H1 (5′-ACCUCUUUGACCUGGACUAT-3′) and SUV39H2 (5′-GAAGCUACCUUUGGUUGUUTT-3′) siRNA oligonucleotides were obtained from Thermo Scientific. SETDB1A (5′-GGAACUGGAGAAGAUGGAUUGUGUA-3′) and SETDB1B (5′-CCGTGAAGCTATGGCTGCCTTAAGA-3′) were from Dharmacon. BRCA1 (5′-GGAACCUGUCUCCACAAAG-3′) siRNAs were synthesized by Invitrogene (UK). Unless otherwise stated, combined HP1α/β siRNA oligonucleotides were used. Combined SETDB1A/B and combined SUV39H1/H2 were used in all experiments.
The pCBASceI (from Dr M Jasin) plasmid was transfected using GeneJuice transfection reagent (Novagen, Germany) per 2.5 × 105 cells at 24 h post siRNA transfection.