Journal: International Journal of Molecular Sciences

Article Title: APE1 Promotes Pancreatic Cancer Proliferation through GFRα1/Src/ERK Axis-Cascade Signaling in Response to GDNF

doi: 10.3390/ijms21103586

Figure Lengend Snippet: APE1 promotes pancreatic cancer cell proliferation via GFRα1. ( A ) MIA PaCa-2 cells were incubated with GDNF for 24 h. Cell proliferation was analyzed by WST-1 assay. The values are represented as a mean ± standard deviation from three independent experiments. ** Denotes p < 0.01 by t -test for equality of means. ( B ) MIA PaCa-2 cells were incubated with or without GDNF (50 ng/mL) for up to 96 h. The number of cells was determined by BrdU assay every 24 h after GDNF treatment. The values are represented as a mean ± standard deviation from three independent experiments. ** Denotes p < 0.01 by t -test for equality of means. ( C and D ) MIA PaCa-2 cells were transfected with control siRNA, APE1 siRNA, or GFRα1 siRNA. After transfection, the cells were treated for 48 h with or without 50 ng/mL GDNF. ( C ) Western blot analysis of APE1 and GFRα1. ( D ) Cell proliferation was analyzed by WST-1 assay. The results indicate the percentage of cell proliferation compared with untreated controls (adjusted to 100%) from three independent experiments. ** Denotes p < 0.01 by t -test for equality of means. ( E and F ) Control siRNA (scrambled) or APE1 siRNA-transfected MIA PaCa-2 cells were cultured and transduced with either control GFP or GFRα1 lentivirus. After transfection, the cells were treated for 48 h with or without 50 ng/mL GDNF. ( E ) Western blot analysis of APE1 and GFRα1. ( F ) Cell proliferation was analyzed by WST-1 assay. The results indicate the percentage of cell proliferation compared with untreated controls (adjusted to 100%) from three independent experiments. ** Denotes p < 0.01 by t -test for equality of means.

Article Snippet: Control siRNA (sc-37007) and c-Src siRNA (sc-29527) were purchased from Santa Cruz Biotechnology as negative controls.

Techniques: Incubation, WST-1 Assay, Standard Deviation, BrdU Staining, Transfection, Control, Western Blot, Cell Culture, Transduction

Journal: International Journal of Molecular Sciences

Article Title: APE1 Promotes Pancreatic Cancer Proliferation through GFRα1/Src/ERK Axis-Cascade Signaling in Response to GDNF

doi: 10.3390/ijms21103586

Figure Lengend Snippet: APE1 and GFRα1 increases ERK phosphorylation in response to GDNF. ( A ) MIA PaCa-2 cells were pretreated with MEK-1 inhibitor PD98059 (10 μM) and the PI3K inhibitor Wortmannin (200 μM) and then treated with or without 50 ng/mL GDNF for 24 h. After treatment, cell proliferation was analyzed by WST-1 assay. Data are expressed as mean ± standard deviation from three independent experiments. ** and # denote p < 0.01 by t -test for equality of means. ( B and C ) MIA PaCa-2 cells were transfected with APE1 siRNA ( B ) or GFRα1 siRNA ( C ) and incubated with GDNF (50 ng/mL). Cells were lysed at the indicated times and subjected to immunoblotting with indicated antibodies.

Article Snippet: Control siRNA (sc-37007) and c-Src siRNA (sc-29527) were purchased from Santa Cruz Biotechnology as negative controls.

Techniques: Phospho-proteomics, WST-1 Assay, Standard Deviation, Transfection, Incubation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: APE1 Promotes Pancreatic Cancer Proliferation through GFRα1/Src/ERK Axis-Cascade Signaling in Response to GDNF

doi: 10.3390/ijms21103586

Figure Lengend Snippet: The role of APE1 on Src/ERK phosphorylation via GDNF/GFRα1 signaling. ( A ) MIA PaCa-2 cells were transfected with control siRNA or Src siRNA. Forty-eight hours after transfection, cells were incubated with GDNF (50 ng/mL) for 1 h. Total cell extracts were prepared for immunoblotting with indicated antibodies. ( B ) Control or Src siRNA-transfected MIA PaCa-2 cells were incubated with or without GDNF (50 ng/mL) for up to 96 h. The number of cells was determined by counting the cells every 24 h after GDNF treatment. The values are represented as a mean ± standard deviation from three independent experiments. ** Denotes p < 0.01 by t -test for equality of means. ( C ) MIA PaCa-2 cells were treated with or without indicated amounts of PP1 and then incubated with GDNF (50 ng/mL) for 1 h. Total cell extracts were prepared for immunoblotting with indicated antibodies. ( D ) MIA PaCa-2 cells were treated with DMSO or PP1 and then incubated with or without GDNF (50 ng/mL) for up to 72 h. The number of cells was determined by BrdU assay every 24 h after GDNF treatment. The values are represented as a mean ± standard deviation from three independent experiments. ** Denotes p < 0.01 by t -test for equality of means. ( E – G ) MIA PaCa-2 cells were transfected with siRNA (control, APE1, or GFRα1), and/or expression vectors (control or GFRα1) and then incubated with GDNF (50 ng/mL) for 1 h. ( E ) Total cell extracts were prepared for immunoblotting with indicated antibodies. ( F ) Relative expression levels of p-Src were quantified by densitometry. The values are represented as a mean ± standard deviation from three independent experiments. ** Denotes p < 0.01 by t -test for equality of means. ( G ) Relative expression levels of pERK1/2 were quantified by densitometry. The values are represented as a mean ± standard deviation from three independent experiments. ** Denotes p < 0.01 by t -test for equality of means.

Article Snippet: Control siRNA (sc-37007) and c-Src siRNA (sc-29527) were purchased from Santa Cruz Biotechnology as negative controls.

Techniques: Phospho-proteomics, Transfection, Control, Incubation, Western Blot, Standard Deviation, BrdU Staining, Expressing

Journal: BMC Cancer

Article Title: Neuropilin-1 promotes the oncogenic Tenascin-C/ integrin β3 pathway and modulates chemoresistance in breast cancer cells

doi: 10.1186/s12885-018-4446-y

Figure Lengend Snippet: Tenascin C contributes to NRP-1 associated migration. a. Dual immunofluorescence staining (40× magnification scale bar 10 μm) of NRP-1 and TNC on BT-474 and BT-474 NRP-1 cells indicates their colocalization in the cytoplasm. Treatment of BT-474 NRP-1 cells with TNC targeted siRNA molecules, b , reduced TNC gene expression, c , reduced migratory capacity and d , downregulated NRP-1 and vimentin expression. (TNC protein was not detected on western blot due to the lack of specific antibody for this application.) The gene expression fold change was measured by comparing the basal levels detected in the empty plasmid transfected BT-474 or in the case of the siRNA experiment, to the control siRNA treated BT-474 and normalized to β-Actin and GUSB reference gene expression. Wound healing assay images (panel d, 5× magnification, scale bar 500 μm) taken on day 0 and day 2 after siRNA transfection. Graphs represent the mean ± SEM of three independent experiments. Statistical analysis using independent samples t-test, p-value < 0.05 considered as statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: Cells were treated with 80 pmol of a pool of three human TNC-targeted siRNA (Santa Cruz) or control siRNA in transfection reagent and serum-free siRNA transfection medium (Santa Cruz) according to the manufacturer’s instructions.

Techniques: Migration, Immunofluorescence, Staining, Gene Expression, Expressing, Western Blot, Plasmid Preparation, Transfection, Control, Wound Healing Assay

Journal: BMC Cancer

Article Title: Neuropilin-1 promotes the oncogenic Tenascin-C/ integrin β3 pathway and modulates chemoresistance in breast cancer cells

doi: 10.1186/s12885-018-4446-y

Figure Lengend Snippet: NRP-1 overexpression activates integrin β3 and TNFR2 pathways. Representative western blot images of protein lysates from untransfected BT-474, BT-474 NRP-1 and empty vector control cells blotted with indicated antibodies involved in a. Integrin signaling, and downstream signaling targets FAK, Akt, GSK3-β and NF-kB b. siRNA-mediated TNC downregulation decreased phosphorylation of FAK and Akt-473. c. Blots show levels of tumor necrosis factor receptors (TNFRs). GAPDH protein expression is indicated as a loading control. (The prefix P beside the antibody names indicates the phosphorylated form)

Article Snippet: Cells were treated with 80 pmol of a pool of three human TNC-targeted siRNA (Santa Cruz) or control siRNA in transfection reagent and serum-free siRNA transfection medium (Santa Cruz) according to the manufacturer’s instructions.

Techniques: Over Expression, Western Blot, Plasmid Preparation, Control, Phospho-proteomics, Expressing