With an altered PKM2 expression were established using RNA interference (PKM

With an altered PKM2 expression were established using RNA interference (PKM2 knockdown) in the BGC823, SGC7901 and AGS cells. As shown in Fig. 1A, the BGC823, SGC7901 and AGS cell lines 10781694 with PKM2 knockdown were established. We observed that the proliferation was decreased in the BGC823 and AGS cells after PKM2 was depleted (Fig. 1B). These results are in agreement with previous studies. To examine the effect of PKM2 on cell migration and invasion, we employed well-established wound-healing and transwell assaysPkM2 Regulates the EGF/EGFR SignalFigure 1. Knockdown of PKM2 promoted the migration and invasion of BGC823 and SGC7901 cells. (A) BGC823, SGC7901 and AGS cells were stably transfected with shRNA directed against PKM2. The specific knockdown of PKM2 was monitored by immunoblot (bottom). Cells stably transfected with pcPUR+U6-siPKM2 are referred to as siPK, and those transfected with pcPUR+U6-siRenilla are referred to as pu6. (B) The proliferation of the stably transfected cells. The cell number was determined with the CCK-8 assay, and the relative number of cells is shown. (C, D) A cross-shaped wound was created in the monolayer, and the BGC823 and SGC7901 stably transfected cells were cultured for an additional 24 hours with EGFPkM2 Regulates the EGF/EGFR Signal(100 ng/ml). Representative images of the wounded region are shown. The results of the migration assay are also shown as graphs (*p,0.05). (E, F) The invasion potential of 10781694 with PKM2 knockdown were established. We observed that the proliferation was decreased in the BGC823 and AGS cells after PKM2 was depleted (Fig. 1B). These results are in agreement with previous studies. To examine the effect of PKM2 on cell migration and invasion, we employed well-established wound-healing and transwell assaysPkM2 Regulates the EGF/EGFR SignalFigure 1. Knockdown of PKM2 promoted the migration and invasion of BGC823 and SGC7901 cells. (A) BGC823, SGC7901 and AGS cells were stably transfected with shRNA directed against PKM2. The specific knockdown of PKM2 was monitored by immunoblot (bottom). Cells stably transfected with pcPUR+U6-siPKM2 are referred to as siPK, and those transfected with pcPUR+U6-siRenilla are referred to as pu6. (B) The proliferation of the stably transfected cells. The cell number was determined with the CCK-8 assay, and the relative number of cells is shown. (C, D) A cross-shaped wound was created in the monolayer, and the BGC823 and SGC7901 stably transfected cells were cultured for an additional 24 hours with EGFPkM2 Regulates the EGF/EGFR Signal(100 ng/ml). Representative images of the wounded region are shown. The results of the migration assay are also shown as graphs (*p,0.05). (E, F) The invasion potential of 18204824 the BGC823 and SGC7901 stable cells was assessed using the BD transwell chamber assay with 100 ng/ml EGF in the lower chamber for 36 hours. The cells that migrated to the lower side of the filter were stained, photographed, and counted. The data are expressed as the mean 6 SD from three independent experiments (*p,0.05). doi:10.1371/journal.pone.0067542.gFigure 2. Depletion of PKM2 decreased the expression of E-cadherin and enhanced the activities of the EGF/EGFR downstream signaling pathways. (A) E-cadherin, phospho-E-cadherin and N-cadherin expression levels were analyzed by immunoblot analysis in BGC823 and SGC7901 stable cells. (B) E-cadherin and N-cadherin expression levels were analyzed by quantitative real-time PCR in BGC823 and SGC7901 stable cells. (C) BGC823 and SGC7901 stable cells were exposed to EGF (100 ng/ml) for different times. The Western blots of cell lysates are shown. The phospho-EGFR (Tyr1068), phospho-PLCc1 (Tyr783), phospho-AKT (ser473), and phospho-ERK1/2 (Thr202/Tyr204) protein levels are shown as indicated. (D) MMP7 expression levels were analyzed by quantitative real-time PCR in BGC823 and SGC7901 stable cells. Error bars represent the mean 6 SD of triplicate experiments (*p,0.05). doi:10.1371/journal.pone.0067542.gPkM2 Regulates the EGF/EGFR SignalDepletion of PKM2 Attenuated the Motility of AGS Cells and the Functional Changes after Rescuing PKM2 in Gastric Cancer Cell LinesThe expression of the E-cadherin protein in the gastric cancer cell lines BGC823, SGC7901 and AGS was evaluated with Western blot analysis. The BGC823 and SGC7901 cell lines express E-cadherin. In contrast, AGS cells lack E-cadherin expression (Fig. 3A). To examine the effect of PKM2 knockdown on cell migration and invasion in AGS cells, we employed well-established woundhealing and transwell assays to characterize the cell motility. A confluent layer of cells was first incubated overnight in medium, a wound scratch was introduced, medium containing EGF (100 ng/ ml) was added to stimulate migration, and t.