Notch2 sirna

Notch2-siRNA (sc-40135, Santa Cruz Biotechnology, Inc) is the target-specific 19-25 nt siRNA designed to knock down human Notch2 gene expression. For siRNA transfection, GC cells were seeded into 6-well plates to be grown to sub-confluency and transiently transfected with negative control siRNA (sc-44236, Santa Cruz Biotechnology, Inc) or Notch2-siRNA at 50 nM for 24 h using Lipofectamine 2000 (Invitrogen, San Diego, CA, USA) according to the manufacturer’s instruction. The next day, the medium was changed to fresh medium with or without ACGs (5 μg/ml), and the cells were continually cultured for 24 h. The proteins were extracted from cells to measure Notch2 expression by western blot and the cell viability was detected by MTS assay.

Cells were incubated with culture medium containing DDP for 24 h after transfection with miR-181b mimics, miR-181b inhibitor, or negative control. For apoptosis analysis, cells were then collected, washed with PBS, resuspended in 100 μL of 1× binding buffer and stained with 5 μL of Annexin V and 5 μL of PI (Becton- Biosciences) at room temperature for 15 min in the dark. A flow cytometer (Becton Biosciences) was utilized to evaluate the levels of apoptosis in each sample following the manufacturer’s instructions.
We assessed the expression of the CD133 surface marker after transfection with miR-181b mimics (Invitrogen) or Notch2 siRNA (Santa Cruz Biotechnology). Briefly, cells were washed with PBS, stained with anti-CD133 (PE-conjugated; Miltenyi Biotec) antibody in PBS containing 1% FBS and incubated on ice in the dark for 30 min. The cells were washed again with cold PBS, and > 10,000 cells were analysed by flow cytometry. The data were analysed using FACS Diva software.