Ration (Figure 3B) and the G1/S transition in NPC 6?0B and HONE1 cells(Figure 3C), compared to their respective Si-Ctrsimilar to the cell migration assay, except that the transwell membranes were pre-coated with 24 mg/ml Matrigel (R D Systems, USA).Examination of CTGF Promoter Methylation by DNA Methylation Microarray AssayThe examination procedure for NimbleGen DNA methylation microarray for 17 NPCs and 3 NP tissues has been described [14], [17]. All experiments were performed at the Kangchen Biology Corporation, Shanghai, China.Statistical AnalysisAll data were analyzed for statistical significance using SPSS 13.0 software. The unpaired T test was applied to test the differential mRNA expression of CTGF in NPC tissues compared to NP tissues. The Chi-square test was used to examine the differences of CTGF protein expression between normal epithelium and cancer tissues of nasopharynx. The Chi-square test was applied to the examination of relationship between CTGF expression levels and clinicopathologic characteristics. One-way ANOVA was used to determine the differences between groupsCTGF in NPCFigure 2. Stable suppression of CTGF expression stimulated the expression of PCNA and sped up cell proliferation, plate clone formation, and cell cycle transition from G1 to S in vitro. A. Stably knocking down CTGF increased the expression of proliferation marker PCNA in shRNA-CTGF-A and B cells compared to inhibitor PLV-Ctr cells by western blot. B. In vitro viability of NPC cells was increased in CTGF-suppressed cells compared to PLV-Ctr cells by CCK8 assay. C. In vitro proliferative ability of NPC cells was significantly increased in CTGF-suppressed cells compared to PLV-Ctr cells by colony formation assay. D. Stably downregulated CTGF expression stimulated cell cycle transition from G1 to S in shRNA-CTGF-A and B cells. One-way ANOVA was used for CCK8 assay, plate clone formation and cell cycle assay. Data were presented as mean6SD for three independent experiments (*p,0.05). doi:10.1371/journal.pone.0064976.gtreated NPC cells. These results suggested a significant inhibitory effect of CTGF on cell growth in vitro.Knock-down of CTGF Facilitates Cell Migration and InvasionTo examine the effect of CTGF on cell migration, stably shRNA-CTGF-expressing 1024 and 1047 6?0B NPC cells were cultured 23148522 on transwell apparatus. After 12-h incubation, theCTGF in NPCFigure 3.Transient suppression of CTGF expression induced the expression of PCNA and promoted cell proliferation, plate clone formation, and cell cycle transition from G1 to S in vitro. A. Suppression of CTGF expression by siRNA induced the expression of PCNA in 6?10B cells and HONE1 cells by western blot. B.Transiently reducing the expression of CTGF by siRNA stimulated cell proliferation in 6?0B cells and HONE1 cells. C. Transiently knocking down the expression of CTGF promoted G1 to S cell cycle transition in NPC 6?0B and HONE cells. One-way ANOVA was used for CCK8 assay and cell cycle assay. Data were presented as mean6SD for three independent experiments (*p,0.05). doi:10.1371/journal.pone.0064976.inhibitor gpercentage of migrated cells in both shRNA-CTGF-1024 and 1047 NPC cell groups was significantly more than that in the PLV-Ctr cells (for both P,0.001) (Figure 4A). Using a boyden chamber coated with matrigel, we determined changes in cell invasiveness after 16 h incubation. Compared with the PLV-Ctr cells, shRNA-CTGF-expressing 1024 and 1047 6?0B NPC cells both showed significantly increased invasiveness (for.Ration (Figure 3B) and the G1/S transition in NPC 6?0B and HONE1 cells(Figure 3C), compared to their respective Si-Ctrsimilar to the cell migration assay, except that the transwell membranes were pre-coated with 24 mg/ml Matrigel (R D Systems, USA).Examination of CTGF Promoter Methylation by DNA Methylation Microarray AssayThe examination procedure for NimbleGen DNA methylation microarray for 17 NPCs and 3 NP tissues has been described [14], [17]. All experiments were performed at the Kangchen Biology Corporation, Shanghai, China.Statistical AnalysisAll data were analyzed for statistical significance using SPSS 13.0 software. The unpaired T test was applied to test the differential mRNA expression of CTGF in NPC tissues compared to NP tissues. The Chi-square test was used to examine the differences of CTGF protein expression between normal epithelium and cancer tissues of nasopharynx. The Chi-square test was applied to the examination of relationship between CTGF expression levels and clinicopathologic characteristics. One-way ANOVA was used to determine the differences between groupsCTGF in NPCFigure 2. Stable suppression of CTGF expression stimulated the expression of PCNA and sped up cell proliferation, plate clone formation, and cell cycle transition from G1 to S in vitro. A. Stably knocking down CTGF increased the expression of proliferation marker PCNA in shRNA-CTGF-A and B cells compared to PLV-Ctr cells by western blot. B. In vitro viability of NPC cells was increased in CTGF-suppressed cells compared to PLV-Ctr cells by CCK8 assay. C. In vitro proliferative ability of NPC cells was significantly increased in CTGF-suppressed cells compared to PLV-Ctr cells by colony formation assay. D. Stably downregulated CTGF expression stimulated cell cycle transition from G1 to S in shRNA-CTGF-A and B cells. One-way ANOVA was used for CCK8 assay, plate clone formation and cell cycle assay. Data were presented as mean6SD for three independent experiments (*p,0.05). doi:10.1371/journal.pone.0064976.gtreated NPC cells. These results suggested a significant inhibitory effect of CTGF on cell growth in vitro.Knock-down of CTGF Facilitates Cell Migration and InvasionTo examine the effect of CTGF on cell migration, stably shRNA-CTGF-expressing 1024 and 1047 6?0B NPC cells were cultured 23148522 on transwell apparatus. After 12-h incubation, theCTGF in NPCFigure 3.Transient suppression of CTGF expression induced the expression of PCNA and promoted cell proliferation, plate clone formation, and cell cycle transition from G1 to S in vitro. A. Suppression of CTGF expression by siRNA induced the expression of PCNA in 6?10B cells and HONE1 cells by western blot. B.Transiently reducing the expression of CTGF by siRNA stimulated cell proliferation in 6?0B cells and HONE1 cells. C. Transiently knocking down the expression of CTGF promoted G1 to S cell cycle transition in NPC 6?0B and HONE cells. One-way ANOVA was used for CCK8 assay and cell cycle assay. Data were presented as mean6SD for three independent experiments (*p,0.05). doi:10.1371/journal.pone.0064976.gpercentage of migrated cells in both shRNA-CTGF-1024 and 1047 NPC cell groups was significantly more than that in the PLV-Ctr cells (for both P,0.001) (Figure 4A). Using a boyden chamber coated with matrigel, we determined changes in cell invasiveness after 16 h incubation. Compared with the PLV-Ctr cells, shRNA-CTGF-expressing 1024 and 1047 6?0B NPC cells both showed significantly increased invasiveness (for.