Rnase h

Cells (300,000 cells/well) were seeded in 6-well plates. MRT and WT cells were treated with 400 nM and 100 nM Selinexor, respectively, for 48 hours. Cells were then fixed in 70% ethanol, washed, and stained in PBS 1X containing 50 μg/ml RNase H (Sigma Aldrich) and 50 μg/ml propidium iodide (PI) (Sigma Aldrich), followed by flow cytometric analysis.

COS7 cells were transfected with the (CGG)₈₈-GFP plasmid and treated with compound followed by isolation of total RNA as described above. After RQ1 DNase treatment, total RNAs were phenol:chloroform extracted and ethanol precipitated. Next, 10 μg of total RNA (100 μL) in RNase H Buffer (20 mM Hepes, pH 7.5, 100 mM KCl, 20 mM MgCl₂, and 0.1 mM DTT) was annealed with 500 pmoles of antisense oligonucleotide at 37 °C with slow cooling to room temperature over 20 min. RNase H (Life Sciences) (5 U) was added, and the mixture was incubated at 37 °C for 30 min. After, the mixture was heated at 65 °C for 20 min to deactivate the RNase H, and then the samples were incubated with streptavidin beads (100 μL, Sigma-Aldrich) for 1 h at room temperature with gentle shaking. The solution was removed, and the beads were washed with 5 × 200 μL H₂O for 5 min each, until the presence of RNA was no longer detected in the wash solution as determined by absorbance at 260 and 280 nm. Bound RNA was released from the beads by heating the beads in 1× Elution Buffer at 65 °C for 5 min. The released RNA was purified by ethanol precipitation, and cDNA was generated from 20 ng of RNA using a qScript cDNA Synthesis Kit per the manufacturer's protocol. qPCR was performed on an ABI 7900 HT Real-Time PCR System. The sequences of antisense oligonucleotide and primers (a-d) are provided in Table S-1.