Journal: American Journal of Cancer Research

Article Title: MiR-182 promotes proliferation and invasion and elevates the HIF-1α-VEGF-A axis in breast cancer cells by targeting FBXW7

doi:

Figure Lengend Snippet: Association of miR-182 with FBXW7 and clinicopathological parameters of breast cancer

Article Snippet: Membranes were blocked with nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBST) and then incubated with primary antibodies against FBXW7, Cyclin E (Santa Cruz, Dallas, TX), Notch (Abcam, Cambridge, UK), FLAG (Sigma, St. Louis, MO), HIF1-α and VEGF-A (Genetex, Irvine, CA) and actin (Millipore, Temecula, CA).

Techniques:

Journal: American Journal of Cancer Research

Article Title: MiR-182 promotes proliferation and invasion and elevates the HIF-1α-VEGF-A axis in breast cancer cells by targeting FBXW7

doi:

Figure Lengend Snippet: FBXW7 was targeted by miR-182. A. FBXW7 mRNA and protein levels were detected in normal mammary epithelial cells and different breast cancer cell lines. B. Overexpression or knockdown of miR-182 could inhibit or increase the protein level of FBXW7 but not the mRNA level. C. The miR-182 targeting sequence in the FBXW7 3’UTR was shown and mutagenesis was performed to change the three nucleotides underlined (upper panel). Wild type or mutated FBXW7 3’UTR reporters were transfected into H184B5F5/M10 and H184B5F5/M10-miR-182 cells and the reporter activities were compared (left panel). In addition, the control reporter vector (pEZX) or FBXW7 3’UTR reporter vector was transfected into H184B5F5/M10-miR-182 cells and the reporter activities were compared (right panel). D. The control reporter vector (pEZX) or FBXW7 3’UTR reporter vector was transfected into MCF-7 or MCF-7 Sponge cells and the reporter activities were compared. ***P<0.001, **P<0.01 and *P<0.05.

Article Snippet: Membranes were blocked with nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBST) and then incubated with primary antibodies against FBXW7, Cyclin E (Santa Cruz, Dallas, TX), Notch (Abcam, Cambridge, UK), FLAG (Sigma, St. Louis, MO), HIF1-α and VEGF-A (Genetex, Irvine, CA) and actin (Millipore, Temecula, CA).

Techniques: Over Expression, Knockdown, Sequencing, Mutagenesis, Transfection, Control, Plasmid Preparation

Journal: American Journal of Cancer Research

Article Title: MiR-182 promotes proliferation and invasion and elevates the HIF-1α-VEGF-A axis in breast cancer cells by targeting FBXW7

doi:

Figure Lengend Snippet: MiR-182 affected the protein level of two FBXW7 degradation substrates cyclin E and Notch. A. The protein level of FBXW7, cyclin E and Notch were investigated in different cell lines. B. Knockdown of FBXW7 by siRNA (FBSi) in H184B5F5/M10 cells or ectopic expression of FBXW7 (FBXW7 expression vector, FBEX) in MCF-7 cells modulated the protein level of cyclin E and Notch.

Article Snippet: Membranes were blocked with nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBST) and then incubated with primary antibodies against FBXW7, Cyclin E (Santa Cruz, Dallas, TX), Notch (Abcam, Cambridge, UK), FLAG (Sigma, St. Louis, MO), HIF1-α and VEGF-A (Genetex, Irvine, CA) and actin (Millipore, Temecula, CA).

Techniques: Knockdown, Expressing, Plasmid Preparation

Journal: American Journal of Cancer Research

Article Title: MiR-182 promotes proliferation and invasion and elevates the HIF-1α-VEGF-A axis in breast cancer cells by targeting FBXW7

doi:

Figure Lengend Snippet: MiR-182 enhanced hypoxia-induced HIF-1α expression and angiogenesis. A. H184B5F5/M10 and H184B5F5/M10-miR-182 cells were cultured in normoxia and 1% O2 for 24 h and the protein level of FBXW7 and HIF-1α was investigated by western blot analysis. B. H184B5F5/M10, H184B5F5/M10-miR-182 and H184B5F5/M10-miR-182 cells transfected with FBXW7 (182-FBXW7) were cultured in normoxia and 1% O2 for 24 h. Total RNAs were isolated and the expression of VEGF-A, -C, and -D was studied by real-time RT-PCR. C. Cells were treated as described in B and the protein level of HIF-1α, FBXW7 and secreted VEGF-A was detected by Western blot analysis. D. H184B5F5/M10 and H184B5F5/M10-miR-182 cells were cultured in 1% O2 for 24 h. The conditioned media were collected and were used to treat endothelial cells (lane 1 and 2). The conditioned media of hypoxia-treated H184B5F5/M10-miR-182 cells were also incubated with control IgG or anti-VEGF-A antibody before treating endothelial cells (lane 3 and 4). The conditioned media of H184B5F5/M10-miR-182 cells transfected with FBXW7 expression vector were collected to treat endothelial cells (lane 5). Results from three independent assays were expressed as Mean ± SE. **P<0.01 and *P<0.05.

Article Snippet: Membranes were blocked with nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBST) and then incubated with primary antibodies against FBXW7, Cyclin E (Santa Cruz, Dallas, TX), Notch (Abcam, Cambridge, UK), FLAG (Sigma, St. Louis, MO), HIF1-α and VEGF-A (Genetex, Irvine, CA) and actin (Millipore, Temecula, CA).

Techniques: Expressing, Cell Culture, Western Blot, Transfection, Isolation, Quantitative RT-PCR, Incubation, Control, Plasmid Preparation