Figure 2. Knockdown of NRDc expression attenuates gastric cancer cell growth.

1. WT TMK-1, MKN-1 and MKN-45 cells were transiently transfected with the control or miR-NRDc plasmids. At 72 h after transfection, 1.5 or 3.0 × 10³ of the cells were seeded in 96-well plates and cultured in the growth medium for 48 h (TMK-1 and MKN-45) or 96 h (MKN-1). The number of viable cells was quantified using the MTS assay (upper panels). Knockdown effects were confirmed by Western blotting (lower panels).
2. The MTS assay was performed using the WT, negative control clone (NC), or stable KD TMK-1 clones (NRDc-KD #1 and #2).
3. The NRDc-KD #1 TMK-1 cells were transfected with the NRDc-FLAG-expressing plasmids at increasing doses. At 48 h after transfection, the MTS assay was performed in the same manner. Recovery of NRDc expression was confirmed by Western blotting (lower panels).
4. Cell lysates were prepared from the indicated clones without using protease inhibitors. TACE activity of each lysate is shown in the upper panel. Lower panels, ADAM17 protein expression was analysed by Western blotting.
5. Left panel, siRNA targeting ADAM17, ADAM10 or the combination was introduced into TMK-1 cells. At 72 h after transfection, qRT-PCR was performed. Right panel, after TMK-1 cells were incubated with the indicated siRNA duplex for 48 h, MTS assay was performed for the additional 48 h as described in Fig 2A.