Journal: Annals of Surgical Oncology

Article Title: Inhibition of p600 Expression Suppresses Both Invasiveness and Anoikis Resistance of Gastric Cancer

doi: 10.1245/s10434-010-1523-0

Figure Lengend Snippet: Inhibition of p600 reduces the invasiveness of gastric cancer cells. a Effect of p600-targeting siRNA in the MKN45 gastric cancer cell line. ( i ) Reverse transcriptase–polymerase chain reaction (RT-PCR). ( ii ) Western blot test. b Effect of p600-targeting siRNA in the AGS gastric cancer cell line. ( i ) RT-PCR. ( ii ) Western blot test. β-Actin was used as the internal control for immunoblotting. p600 expression in both gastric cancer cell lines was inhibited by p600-targeting siRNA for at least 5 days. c ( i ) Microscopic appearance of the invading control siRNA-transfected MKN45 cells, and ( ii ) p600-targeting siRNA-transfected MKN45 cells . ( iii ) Number of invaded cells. d ( i ) Microscopic appearance of invading control siRNA-transfected AGS cells, and ( ii ) p600-targeting siRNA-transfected AGS cells. ( iii ) Number of invaded cells. Data are expressed as mean ± SD. Inhibition of p600 reduced the invasiveness of both gastric cancer cell lines (MKN45, P = 0.004; AGS, P < 0.001). e Western blot analysis of urokinase-type plasminogen activator (uPA), matrix metalloproteinase (MMP)-2, MMP-7, and MMP-9 expression levels. ( i ) MKN45. ( ii ) AGS. β-Actin was used as the internal control for immunoblotting. MMP-7 expression was decreased by inhibition of p600 in both gastric cancer cell lines. f Western blot analysis of E-cadherin expression in MKN45 gastric cancer cells. β-Actin was used as the internal control for immunoblotting. Expression of E-cadherin was higher in p600-targeting siRNA-transfected MKN45 cells than in control siRNA-transfected MKN45 cells

Article Snippet: Cells were transfected with p600-targeting small interfering RNA (siRNA) (sense, ggaaagaacaucauuguuaTT; antisense, uaacaaugauguucuuuccTT) (B-Bridge, Mountain View, CA), or negative control siRNA (B-Bridge), which was confirmed not to target a specific array by electroporation with a Nucleofector System (Amaxa Biosystems, Koln, Germany) according to the manufacturer’s instructions.

Techniques: Inhibition, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Western Blot, Expressing, Transfection

Journal: Annals of Surgical Oncology

Article Title: Inhibition of p600 Expression Suppresses Both Invasiveness and Anoikis Resistance of Gastric Cancer

doi: 10.1245/s10434-010-1523-0

Figure Lengend Snippet: Inhibition of p600 does not affect cell proliferation, apoptosis, and colony-formation capacity under anchorage-dependent conditions. a Proliferation assay under anchorage-dependent conditions. There was no significant difference between p600 -targeting siRNA-transfected MKN45 cells and control siRNA-transfected MKN45 cells in terms of cell proliferation under anchorage-dependent conditions. b Proportion of apoptotic cells in ( i ) control siRNA-transfected MKN45 cells and ( ii ) p600-targeting siRNA-transfected MKN45 cells as analyzed by flow cytometry. There was no significant difference between p600-targeting siRNA-transfected MKN45 cells and control siRNA-transfected MKN45 cells in terms of apoptotic cells proportion. c Macroscopic appearance of colonies formed under anchorage-dependent conditions; ( i ) control siRNA-transfected MKN45 cells and ( ii ) p600 - targeting siRNA-transfected MKN45 cells. ( iii ) Data graph. Data are expressed as mean ± SD. There was no significant difference between p600 -targeting siRNA-transfected MKN45 cells and control siRNA-transfected MKN45 cells in terms of colony-formation capacity under anchorage-dependent conditions

Article Snippet: Cells were transfected with p600-targeting small interfering RNA (siRNA) (sense, ggaaagaacaucauuguuaTT; antisense, uaacaaugauguucuuuccTT) (B-Bridge, Mountain View, CA), or negative control siRNA (B-Bridge), which was confirmed not to target a specific array by electroporation with a Nucleofector System (Amaxa Biosystems, Koln, Germany) according to the manufacturer’s instructions.

Techniques: Inhibition, Proliferation Assay, Transfection, Flow Cytometry

Journal: Annals of Surgical Oncology

Article Title: Inhibition of p600 Expression Suppresses Both Invasiveness and Anoikis Resistance of Gastric Cancer

doi: 10.1245/s10434-010-1523-0

Figure Lengend Snippet: Inhibition of p600 sensitizes gastric cancer cells to anoikis and reduces their viability under anchorage-independent conditions. a Cell viability assay under anchorage-independent conditions. The viability of ( i ) MKN45 and ( ii ) AGS gastric cancer cells were reduced by inhibition of p600 (MKN45, P = 0.033; AGS, P < 0.001). b Proportion of apoptotic (anoikis) cells under anchorage-independent conditions in ( i ) control siRNA-transfected MKN45 cells and ( ii ) p600-targeting siRNA-transfected MKN45 cells as analyzed by flow cytometry. c Proportion of apoptotic (anoikis) cells in ( i ) control siRNA-transfected AGS cells and ( ii ) p600-targeting siRNA-transfected AGS cells as analyzed by flow cytometry. d Macroscopic appearance of colonies formed in soft agar; ( i ) control siRNA-transfected MKN45 cells and ( ii ) p600-targeting siRNA-transfected MKN45 cells. ( iii ) Number of colonies. e Macroscopic appearance of colonies formed in soft agar; ( i ) control siRNA-transfected AGS cells and ( ii ) p600-targeting siRNA-transfected AGS cells. ( iii ) Data graph. Data are expressed as mean ± SD. The number of colonies was reduced by inhibition of p600 (MKN45, P < 0.001; AGS, P = 0.003)

Article Snippet: Cells were transfected with p600-targeting small interfering RNA (siRNA) (sense, ggaaagaacaucauuguuaTT; antisense, uaacaaugauguucuuuccTT) (B-Bridge, Mountain View, CA), or negative control siRNA (B-Bridge), which was confirmed not to target a specific array by electroporation with a Nucleofector System (Amaxa Biosystems, Koln, Germany) according to the manufacturer’s instructions.

Techniques: Inhibition, Viability Assay, Transfection, Flow Cytometry

Journal: Annals of Surgical Oncology

Article Title: Inhibition of p600 Expression Suppresses Both Invasiveness and Anoikis Resistance of Gastric Cancer

doi: 10.1245/s10434-010-1523-0

Figure Lengend Snippet: Inhibition of p600 suppresses the establishment of intraperitoneal disseminated tumors. a Macroscopic appearance of the intraperitoneal disseminated nodules; ( i ) control siRNA-transfected MKN45 cells injected into SCID mice, and ( ii ) p600-targeting siRNA-transfected MKN45 cells ( n = 10/group). Arrows indicate intraperitoneal disseminated nodules. ( iii ) Resected disseminated nodules from SCID mice. b Data graph. Data are expressed as mean ± SD. The number of intraperitoneal disseminated nodules was significantly reduced by p600 inhibition ( P < 0.001)

Article Snippet: Cells were transfected with p600-targeting small interfering RNA (siRNA) (sense, ggaaagaacaucauuguuaTT; antisense, uaacaaugauguucuuuccTT) (B-Bridge, Mountain View, CA), or negative control siRNA (B-Bridge), which was confirmed not to target a specific array by electroporation with a Nucleofector System (Amaxa Biosystems, Koln, Germany) according to the manufacturer’s instructions.

Techniques: Inhibition, Transfection, Injection

Journal: PLoS ONE

Article Title: A Role for Methyl-CpG Binding Domain Protein 2 in the Modulation of the Estrogen Response of pS2 / TFF1 Gene

doi: 10.1371/journal.pone.0009665

Figure Lengend Snippet: Real time RT-PCR analysis of pS2 transcripts in HeLa and MBD2 -depleted HeLa cells (HeLa cells pretreated for 72 h with MBD2 siRNA) transfected with an MBD2 vector expressing a transcript resistant to RNAi (pRev-MBD2 vector) or with an empty basic vector pGL3. Transcriptional expression of pS2 was analyzed 24 h after transfection. The fold change of pS2 expression was calculated from the relative pS2 mRNA in pRev-MBD2-transfected cells compared to pGL3-transfected cells. Values are presented as the mean ± standard deviation of at least three independent transfection experiments. A significant difference between the two cell groups is represented by an asterisk * (P<0.05).

Article Snippet: siRNA duplexes for MBD2 (sense: 5′-GGAGGAAGUGUACCGAAATT-3′ ; antisense: 5′-UUUUCGGAUCACUUCCUCCTT-3′ ) and non-specific siRNA control were obtained from Eurogentec (Eurogentec, Seraing, Belgium).

Techniques: Quantitative RT-PCR, Transfection, Plasmid Preparation, Expressing, Standard Deviation

Journal: PLoS ONE

Article Title: A Role for Methyl-CpG Binding Domain Protein 2 in the Modulation of the Estrogen Response of pS2 / TFF1 Gene

doi: 10.1371/journal.pone.0009665

Figure Lengend Snippet: ( A ) Transcriptional expression of pS2 was analyzed using relative RT-PCR in HeLa cells expressing ERα and/or depleted in MBD2. Mock, mock-treated HeLa cells. ER, HeLa cells 24 h after transfection with a human ERα expression vector, HEG0. MBD2 siRNA, HeLa cells pretreated for 72 h with MBD2 siRNA and again for 24 h. MBD2 siRNA + ER, HeLa cells pretreated for 72 h with MBD2 siRNA, then cotransfected with MBD2 siRNA and HEG0 for 24 h. OHT, 24 h treatment with 4-hydroxytamoxifen. MCF7, MCF7 cells. ( B ) Bar chart showing the fold change of pS2 expression in HeLa cells expressing ERα and/or depleted in MBD2. pS2 transcripts were quantified by real time RT-PCR. The fold change was calculated from the relative pS2 mRNA in treated compared to mock-treated cells. Each bar represents the mean ± standard deviation of three analyses. A significant difference between the two cell groups is represented by an asterisk * (P<0.05).

Article Snippet: siRNA duplexes for MBD2 (sense: 5′-GGAGGAAGUGUACCGAAATT-3′ ; antisense: 5′-UUUUCGGAUCACUUCCUCCTT-3′ ) and non-specific siRNA control were obtained from Eurogentec (Eurogentec, Seraing, Belgium).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation, Quantitative RT-PCR, Standard Deviation