Fig 1.
Overexpression of DPY30 in gastric cancers.
(A-D) Immunohistochemical staining demonstrated the overexpression of DPY30 in gastric cancer tissues. Notably its overexpression was obviously greater in invading cancer cells (B-D) than in normal gastric mucosa (A). (E) The mRNA level of DPY30 in gastric cancer cells (SNU1, SNU16, SNU216, SNU620, SNU638 and NCI-N87) and normal gastric epithelial cell (HFE145) was determined by real-time PCR using specific primers for DPY30. GAPDH was used to normalize data. Values shown are the means ± SDs of the three independent experiments performed in triplicate. *, p < 0.01 (Student’s t test, versus HFE145). (F) The expression of DPY30 in gastric cancer tissues was examined by real-time PCR using specific primers for DPY30. GAPDH was used to normalize data. Values are the means ± SDs of three independent experiments performed in triplicate. *, p < 0.01; **, p < 0.05 (Student’s t test, versus normal).
Fig 2.
DPY30 regulated the proliferation of gastric cancer cells.
(A) Real-time PCR was used to determine the efficiency of knockdown or overexpression of DPY30 in HFE145, SNU1, SNU16, SNU216 and SNU638 cells. Knockdown efficiency was determined after transfecting cells with 100 nM DPY30 siRNA targeting the ORF or scrambled siRNA (SCR). Overexpression efficiencies were determined in DPY30-overexpressing and mock cells. (B) Effect of DPY30 knockdown or overexpression on cell proliferation. A cell viability assay was used to measure cell proliferation in the presence of 1% FBS. For the DPY30 knockdown experiments, cell viability assays were performed five days after transfecting 100 nM DPY30 siRNA or SCR. After three days of culture, cell viability assays were performed on DPY30-over and mock cells. (C) Expression analysis of DPY30 after known-down or overexpression by siRNAs or DPY30 ORF respectively. The ORF-targeting or the 3’-UTR-targeting siRNA was used for the knock-down. (D) Exogenous DPY30-ORF rescued the inhibition of proliferation by the 3’-UTR-targeting DPY30 siRNA. Mock or DPY30-overexpressing cells were transduced with two kinds of DPY30 siRNA (3’-UTR-targeting or ORF-targeting), then cell viability assay was examined. Values are the means ± SDs of three independent experiments performed in triplicate. *, p < 0.01 (Student’s t test, versus SCR or Mock).
Fig 3.
WRAD components regulated proliferation of gastric cancer cells.
(A) The mRNA levels of WDR5, RBBP5 and ASH2L in HFE145, SNU1, SNU16, SNU216 and SNU638 cells were determined by real-time PCR, using specific primers for WDR5, RBBP5 and ASH2L. GAPDH was used to normalize data. (B) Knockdown efficiency was determined by real-time PCR. Knockdown efficiency was determined after transfecting cells with 100 nM WDR5, RBBP5 and ASH2L siRNA or scrambled (SCR). (C) Effects of WDR5, RBBP5 and ASH2L knockdown on cell proliferation. A cell viability assay was used to measure cell proliferation in the presence of 1% FBS. 24 hrs after transduction, cell viability assays were performed. Values are the means ± SDs of three independent experiments performed in triplicate. *, p < 0.01 (Student’s t test, versus HFE145 (A) or SCR (B-C)).
Fig 4.
DPY30 regulated the migration of gastric cancer cells.
(A) A Boyden chamber assay was used to measure the migration of gastric cancer cells. 10% FBS was used to induce migration, and mitomycin C (0.01 μg/ml) was added to remove the effects of proliferation. Two days after transfection with 100 nM ORF-targeting DPY30 siRNA or scrambled (SCR) siRNA, migration assays (Boyden chamber assay) were performed. Migration assays were carried out on DPY30-over and mock cells after culture for one day. Bar = 100 μm. (B) Migrated cells were counted and results are presented as a bar graph. Values are the means ± SDs of three independent experiments performed in triplicate. *, p < 0.01 (Student’s t test, versus SCR or Mock).
Fig 5.
DPY30 regulated the invasion of gastric cancer cells.
(A-B) A Matrigel invasion assay was used to measure gastric cancer cell invasion. Presented results are representative of the results obtained. 10% FBS was used to induce invasion, and mitomycin C (0.01 μg/ml) was added to remove effects of proliferation. Invasion assays were performed two days after transfection with 100 nM ORF-targeting DPY30 or SCR siRNA. After culture for one day, invasion assay was carried out for the DPY30-over and mock cells. Bar = 100 μm. (C) Invasive cells were counted and results are displayed as a bar graph. Values are the means ± SDs of three independent experiments performed in triplicate. *, p < 0.01 (Student’s t test, versus SCR or Mock).