Journal: Cardiovascular research

Article Title: HIF-1-mediated up-regulation of cardiotrophin-1 is involved in the survival response of cardiomyocytes to hypoxia.

doi: 10.1093/cvr/cvr202

Figure Lengend Snippet: Figure 6 CT-1 inhibition increased hypoxia-induced apoptosis in HL-1. Cells were infected with control siRNA lentiviral particles (siControl) or siRNA lentiviral particles against CT-1 (siCT-1) and exposed to normoxia or hypoxia for 16 h. (A) Analysis of apoptosis by flow cytometry after 7-Amino-Actinomycin (AAD)/PE-Annexin V staining. The flow cytometry profile of one representative experiment is shown. Data were gated for damaged cells (PE-Annexin V2 and 7-AAD+, top left quadrant), necrotic cells or late apoptosis (PE-Annexin V+ and 7-AAD+, top right quadrant), viable cells (PE-Annexin V2 and 7-AAD2, bottom left quadrant), and early apoptotic cells (PE-Annexin V+ and 7-AAD2, bottom right quadrant). (B) Bar graph showing the percentage of early apoptotic cells determined by flow cytometry. Non-infected cells (C). n ¼ 3. *P , 0.05 vs. cells transfected with the same siRNA and exposed to normoxia; #P , 0.05 vs. cells exposed to the same treatment but transfected with siControl. (C) Representative fluorescence microscope images for PE-Annexin V staining (red fluorescence) and nuclear staining (DAPI, blue fluorescence). (D) Caspase 3/7 activity was analysed as described in the Methods section. n ¼ 3, in triplicate. *P , 0.05 vs. cells transfected with the same siRNA and exposed to normoxia; #P , 0.05 vs. cells exposed to the same treatment but transfected with siControl.

Article Snippet: HL-1 cells were transfected with siRNAs using Lipofectamine-RNAiMAX (Invitrogen), as discussed previously.27 After transfection, cells were kept in complete medium for 24 h, arrested, and then exposed to hypoxia for 16 h. In additional experiments, HL-1 cells were infected with siRNA lentiviral particles, siCT-1 (CT-1 shRNA-mouse Lentiviral Particles, sc-39328-V) or siControl (Control shRNA Lentiviral Particles, sc-108080) from Santa Cruz, grown in complete medium for 48 h and selected with puromycin.

Techniques: Inhibition, Infection, Control, Cytometry, Staining, Transfection, Microscopy, Activity Assay

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