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: Free radical biology & medicine

Article Title: Peroxiredoxin 6 (Prdx6) supports NADPH oxidase1 (Nox1)-based superoxide generation and cell migration.

doi: 10.1016/j.freeradbiomed.2016.04.009

Figure Lengend Snippet: Fig. 1. Prdx6 associates with the SH3 domain of Noxa1 and stabilizes its expression. (A) Interaction of the Noxa1 C-terminal SH3 domain (res. 403-476 constructed in pGBKT7) with Prdx6 was detected by yeast two-hybrid screening of a human kidney cDNA library (expressed in pGADT7). Co-transfectants exhibited Gal4-based transcriptional activation, enabling survival on His-/Leu-/Ade-deficient selective medium. (B) Full-length Prdx6 associates with the Nox1 NADPH oxidase complex. Nox1, Noxo1, and Noxa1 encoding plasmids (0.2 ug each) were co- transfected with or without HA-Prdx6 (0.6 ug) in HEK-293 cells. Cleared cell lysates were incubated with anti-p22phox antibody to precipitate Nox1 complexes and associated HA-Prdx6, detected by Western blotting with anti-HA antibody. (C). Overexpressed Prdx6 enhances production of WT Noxa1 but not the SH3 domain mutant form of Noxa1 (W436R). CHO-K1 cells were co-transfected with Noxa1 plasmid (0.3 ug) and different amounts of Prdx6 plasmid (0, 0.3, 0.6, 0.9 ug) for 48 h. Production of HA-Prdx6 and Noxa1 was analyzed in cell lysates by Western blotting with anti-HA or anti-Noxa1 antibody (left). Prdx6 associates with WT Noxa1 but not Noxa1 W436R. The cleared transfected (48 h) CHO-K1 cell lysates were harvested and incubated with anti-HA antibody to precipitate Prdx6 and associated Noxa1 (right). (D) Overexpression of HA-Prdx6 enhances detection of Noxa1 but not Noxa1 W436R in HEK-293 cells. Nox1, Noxo1, and Noxa1 (0.05 ug each) were co-transfected with HA-Prdx6 (0, 0.05, 0.1, or 0.4 ug) and the cells were processed as described in C. (E). Prdx6 associates with WT Noxa1 but not with Noxa1 W436R in HEK-293 cells. Noxa1 (0.1 ug) was co-transfected with or without Prdx6 (0.8 ug) in HEK-293 cells. Products of HA-Prdx6 and Myc-Noxa1 were analyzed in cell lysates harvested 48 h later by Western blotting (left). Cleared lysates were also incubated with anti-Myc antibody to pull down Noxa1 and bound HA-Prdx6 detected by Western blotting (right).

Article Snippet: Rabbit polyclonal Ab against Noxa1 (gift from Miklos Geiszt, Semmelweis University) and Mouse monoclonal Ab against p22phox (no. 449) were described previously [9]. pRS vector (#TR20003), GFP-specific shRNA vector (#TR30003), and four human Prx6-targeted pRS shRNA expression vectors (#TR310191) were obtained from OriGene (OriGene Technologies, Inc., Rockville, MD).

Techniques: Expressing, Construct, Two Hybrid Screening, cDNA Library Assay, Activation Assay, Transfection, Incubation, Western Blot, Mutagenesis, Plasmid Preparation, Over Expression