FIGURE 2.

Effect of NRN1 on esophageal cancer (EC) cell proliferation, apoptosis, cell cycle, invasion, and migration. A, Flow cytometry results show induction of apoptosis by re‐expression of NRN1 in KYSE30 and KYSE150 cells, while reduction of apoptosis was found after knockdown of NRN1 in KYSE450 cells. **P < .01. B, Growth curves represent cell viability analyzed by the MTT assay in NRN1 re‐expressed and unexpressed KYSE30 and KYSE150 cells, as well as in KYSE450 cell before and after knockdown of NRN1. Each experiment was repeated in triplicate. **P < .01, ***P < .001. C, Colony formation results show that colony numbers were reduced by re‐expression of NRN1 in KYSE30 and KYSE150 cells, while they were increased by knockdown of NRN1 in KYSE450 cells. Each experiment was repeated in triplicate. Average number of tumor clones is represented by bar diagram. **P < .01, ***P < .001. D, Cell phase distribution in NRN1 unexpressed and re‐expressed KYSE30 and KYSE150 cells, as well as cell phase distribution before and after knockdown of NRN1 in KYSE450 cells. Each experiment was repeated 3 times. *P < .05, **P < .01. E, The migration assays show migration cells before and after restoration of NRN1 expression in KYSE30 and KYSE150 cells. The invasion assays show invasive cells before and after restoration of NRN1 expression in KYSE450. ***P < .001. F, Western blots show the effects of knockdown of NRN1 by different siRNA. NC: siRNA negative control; siNRN1: siRNA for NRN1. G, Western blots show the effects of NRN1 on the levels of caspase‐3, cleaved caspase‐3, Bcl‐2, cyclinA2, cyclinD1, cyclin E, MMP2, MMP7, and MMP9 expression in KYSE30, KYSE150, and KYSE450 cells. GFP: control vector, NRN1: NRN1 expressing vector, β‐actin: internal control. NC: siRNA negative control; siNRN1: siRNA for NRN1