Journal: International journal of oncology

Article Title: ACK1 promotes hepatocellular carcinoma progression via downregulating WWOX and activating AKT signaling.

doi: 10.3892/ijo.2015.2910

Figure Lengend Snippet: Figure 6. The effect of ACK1 knockdown on MHCC-97H cells. (A) Representative images show the migration and invasion ability of MHCC-97H cells transfected with ACK1-shRNA or Control-shRNA (x200). (B) Data are presented as mean relative numbers of invaded or migrated cells from 5 fields (*P<0.01). (C) MHCC-97H cells transfected with ACK1-shRNA or Control-shRNA, respectively, were subjected to western blotting for ACK1, p-ACK1, WWOX, AKT, p-AKT, MMP2 and MMP9 (P<0.01).

Article Snippet: The ACK1 shRNA and scrambled shRNA vector pRS were purchased from OriGene Technologies Inc. (Rockville, MD, USA).

Techniques: Knockdown, Migration, Transfection, shRNA, Control, Western Blot

Journal: International journal of oncology

Article Title: ACK1 promotes hepatocellular carcinoma progression via downregulating WWOX and activating AKT signaling.

doi: 10.3892/ijo.2015.2910

Figure Lengend Snippet: Figure 5. ACK1 regulates apoptosis and proliferation in MHCC-97H cells. (A) Cell proliferation as measured by BrdU incorporation was inhibited by ACK1- shRNA in MHCC-97H cells (n=3, *P<0.01). (B) As assessed by MTT assay, ACK1 knockdown was found to reduce the viability of MHCC-97H cells (n=3, *P<0.01). (C) The activity of the pro-apoptotic caspases 3 and 7 was upregulated after ACK1 knockdown in MHCC-97H cells (n=3, *P<0.01). (D) Quantification of the apoptotic cell population by flow cytometry. ACK1 knockdown MHCC-97H cells were composed of a larger subset of apoptotic cells compared with the control group (n=3, *P<0.01). Values are depicted as mean ± SEM.

Article Snippet: The ACK1 shRNA and scrambled shRNA vector pRS were purchased from OriGene Technologies Inc. (Rockville, MD, USA).

Techniques: BrdU Incorporation Assay, shRNA, MTT Assay, Knockdown, Activity Assay, Flow Cytometry, Control

Journal: Oncogene

Article Title: Ras association domain family member 10 suppresses gastric cancer growth by cooperating with GSTP1 to regulate JNK/c-Jun/AP-1 pathway.

doi: 10.1038/onc.2015.300

Figure Lengend Snippet: Figure 3. Effect of RASSF10 on cell growth and cell migration/invasion. (a) Overexpression of RASSF10 in AGS and MKN45 cells after stable transfection with RASSF10 expression vectors was confirmed by western blotting. (b) MTS viability assays showed RASSF10 expression significantly suppressed growth of AGS and MKN45 cells. (c) Colony-formation ability of AGS and MKN45 cells was significantly inhibited by RASSF10 expression. (d1) RASSF10 expression was knocked down in GES-1 cells by transfection of shRNA against RASSF10. (d2) Cell growth of GES-1 was significantly increased after knockdown of RASSF10. (e) Cell migration ability was significantly inhibited by RASSF10 expression in AGS cells as indicated by wound-healing assay. (f) Cell invasiveness was significantly reduced by RASSF10 expression in AGS and MKN45 cells as indicated by matrigel invasion assay.

Article Snippet: Knockdown of gene expression by shRNA or siRNA Vectors carrying shRNA specifically targeting RASSF10 (shRASSF10) or scrambled control shRNA (shCtrl) were purchased from OriGene Technologies (Rockville, MD, USA).

Techniques: Migration, Over Expression, Stable Transfection, Expressing, Western Blot, Transfection, shRNA, Knockdown, Wound Healing Assay, Invasion Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Dual-specificity phosphatase 14 (DUSP14/MKP6) negatively regulates TCR signaling by inhibiting TAB1 activation.

doi: 10.4049/jimmunol.1300989

Figure Lengend Snippet: FIGURE 6. The enhanced JNK and IKK activa- tion of DUSP14-deficient T cells is attenuated by TAB1 knockdown. (A) Flow cytometry analysis of TAB1 expression in TAB1 shRNA-transfected cells. Murine primary T cells were transfected with GFP-tagged TAB1 shRNAs or a scrambled GFP- tagged shRNA for 36 h, followed by intracellular staining for TAB1. The GFP-tagged shRNA-trans- fected cells were GFP gated for flow cytometry analysis. Samples were stained with the secondary Ab only as negative controls (shaded graph). (B and C) WT and DUSP14-deficient (DUSP14-KO) T cells were transfected with GFP-tagged TAB1 shRNAs or a scrambled GFP-tagged shRNA for 36 h and then stimulated with anti-CD3 Ab for 15 min. The phosphorylation of JNK (B) and IKK (C) in shRNA-transfected cells (GFP gated) was ex- amined by intracellular staining. Data (mean 6 SEM) are representative of three independent ex- periments. *p , 0.05, two-tailed t test.

Article Snippet: Murine primary T cells were transfected with a mixture of four unique 29-mer GFP-tagged TAB1 short hairpin RNAs (shRNAs) or a scrambled GFP-tagged shRNA (both from OriGene Technologies) for 36 h. Cells were stimulated with biotinconjugated anti-CD3 (3 mg/ml; 500A2; eBioscience) Ab plus streptavidin (3 mg/ml; Sigma) for 15 min, followed by intracellular staining for p-JNK (Cell Signaling), p-IKK (Santa Cruz), or p-ERK (Cell Signaling).

Techniques: Knockdown, Flow Cytometry, Expressing, shRNA, Transfection, Staining, Cytometry, Phospho-proteomics, Two Tailed Test

Journal: Aging cell

Article Title: A role for the Werner syndrome protein in epigenetic inactivation of the pluripotency factor Oct4.

doi: 10.1111/j.1474-9726.2010.00585.x

Figure Lengend Snippet: Fig. 2 shRNA-mediated knockdown of WRNp in NCCIT cells. NCCIT cells were transfected with plasmids encoding one of two types of shRNA against WRN, or shRNA against G9a, or one of two control plasmids (CV – empty vector, CS – vector control expressing a scrambled, nontargeting sequence, G()) – G9a-deficient clone, 31–34, 61, and 62 – WRNp-deficient clones, see Table S3). Transfected cells were selected in puromycin, and individual clones were isolated and subjected to western blotting analysis with a WRNp antibody or a c-tubulin control.

Article Snippet: An empty control plasmid (TI20003, Origene) and control nontargeting shRNA (TI30003, Origene) were utilized.

Techniques: shRNA, Knockdown, Transfection, Control, Plasmid Preparation, Expressing, Sequencing, Clone Assay, Isolation, Western Blot

Journal: Aging cell

Article Title: A role for the Werner syndrome protein in epigenetic inactivation of the pluripotency factor Oct4.

doi: 10.1111/j.1474-9726.2010.00585.x

Figure Lengend Snippet: Fig. 8 Expression of stem cell markers in WRNp-deficient cells. (A) Control (CV, CS) and WRNp-deficient cells (31) were treated with retinoic acid (RA) for 7 days. Cells were then harvested, and the presence of stem cell markers was analyzed by western blotting (+, cells treated with RA; ), untreated control samples). TRA-1-60, TRA-1-81, and EpCAM bands are indicated. c-tubulin served as a loading control. (B) WRNp-deficient cells (31) were treated with RA for 7 days. Cells were then transfected with Oct4 siRNA (Oi) or control siRNA (Ci). Three days after transfection, cells were harvested and the expression of stem cell markers analyzed as described above ()RA – untreated cells, +RA – RA-treated cells).

Article Snippet: An empty control plasmid (TI20003, Origene) and control nontargeting shRNA (TI30003, Origene) were utilized.

Techniques: Expressing, Control, Western Blot, Transfection

Journal: Journal of cell science

Article Title: Aminopeptidase P3, a new member of the TNF-TNFR2 signaling complex, induces phosphorylation of JNK1 and JNK2.

doi: 10.1242/jcs.149385

Figure Lengend Snippet: Fig. 6. Phosphorylation of JNK and IkB is induced by APP3 during TNFR1 or TNFR2 signaling. (A) HEK293T-TNFR2 cells transfected with APP3m or APP3c, or APP3 shRNA, were prepared. To inhibit aminopeptidase P enzymatic activity, HEK293T-TNFR2 cells were pre-treated with 100 mM apstatin. Untransfected cells were used as a reference. These cells were stimulated with R2-7 (100 ng/ml) for the indicated time. After cell lysis, western blotting was performed to confirm expression of APP3 and TNFR2, as well as phosphorylation of JNK (P-JNK) JNK1 and JNK2 are indicated by the open arrow and filled arrow, respectively. b-actin served as a loading control. (B) IkB and phospho-IkB were also detected in each HEK293T-TNFR2 cell line by western blotting. (C) HEK293T cells transfected with APP3m or APP3c, or APP3 shRNA, and pre-treated with 100 mM apstatin were stimulated by TNF (100 ng/ml) for the indicated time. After cell lysis, JNK and phospho-JNK (JNK1, open arrow; JNK2, filled arrow) were detected by western blotting. (D) IkB and phospho-IkB were also detected in each HEK293T cell line.

Article Snippet: Two APP3 shRNA plasmids, each with a different target sequence (APP3 shRNA A, 59-AGACCAGACAGTGGTTGTGCTCTCCAACC39; and B, 59-GATGGAGGTTGTGAGTCTTCCTGCTATGT-39), and a scrambled negative control shRNA plasmid (OriGene) were transfected into the cells by lipofection.

Techniques: Phospho-proteomics, Transfection, shRNA, Activity Assay, Lysis, Western Blot, Expressing, Control

Journal: Journal of cell science

Article Title: Aminopeptidase P3, a new member of the TNF-TNFR2 signaling complex, induces phosphorylation of JNK1 and JNK2.

doi: 10.1242/jcs.149385

Figure Lengend Snippet: Fig. 8. Induction of TNF-induced cell death by APP3 knockdown. (A) HEK293Tand HEK293T-TNFR2 cells cultured in a 96-well plate were treated with serially diluted TNF. After 24 hours, cell viability was measured by a WST-8 assay. Results are mean6s.d., n53. (B) HEK293T-TNFR2 cells were transfected with APP3 shRNA A, APP3 shRNA B or scrambled shRNA, and incubated for 48 hours. After cell lysis, suppression of APP3 was confirmed by western blotting. b-actin served as a loading control. (C) HEK293T-TNFR2 cells were transfected with APP3 shRNA A, APP3 shRNA B or scrambled shRNA. After 48 hours, these cells were treated with TNF (100 ng/ml) for 24 or 48 hours. Mock transfected cells were used as a reference. Cell viability was measured by a WST-8 assay. (D) HEK293T-TNFR2 cells pretreated with 100 mM apstatin were treated with TNF (100 ng/ml) for 24 or 48 hours. Cell viability was measured by WST-8 assay. Results are mean6s.d., n53.

Article Snippet: Two APP3 shRNA plasmids, each with a different target sequence (APP3 shRNA A, 59-AGACCAGACAGTGGTTGTGCTCTCCAACC39; and B, 59-GATGGAGGTTGTGAGTCTTCCTGCTATGT-39), and a scrambled negative control shRNA plasmid (OriGene) were transfected into the cells by lipofection.

Techniques: Knockdown, Cell Culture, Transfection, shRNA, Incubation, Lysis, Western Blot, Control