Journal: Oncotarget

Article Title: MicroRNA-432 functions as a tumor suppressor gene through targeting E2F3 and AXL in lung adenocarcinoma

doi: 10.18632/oncotarget.7884

Figure Lengend Snippet: ( A and B ) Predicted miR-432 target sequences in the 3′UTRs of E2F3 and AXL ( W ). The miR-432 mutant ( M ) contained four altered nucleotides in the seed sequence. A549 ( C ) and H1299 ( D ) cells transfected with miR-432 mimics or miR-432 inhibitors, and the expression levels of E2F3(left) and AXL(right) were detected by Western blotting. GAPDH was used as a control. The quantification of each protein band in the result of Western blotting was done using LAS-3000 with MultiGauge software (Fuji film). HEK-293T cells were co-transfected with miR-432 mimics or mimics control with WT/Mut 3′-UTR of E2F3 ( E ) and AXL ( F ). Relative luciferase activity was evaluated. Experiments were performed in triplicate. ( G and H ) The same assay above was also performed in A549. * p < 0.05 compared with mimis control. After co-transfection with miR-432 inhibitor or its control and WT/Mut 3′-UTR of E2F3 ( I ) and AXL ( J ) in H1299, the luciferase activity was detected. * p < 0.05 compared with inhibitor control.

Article Snippet: The slides were incubated with rabbit polyclonal anti-E2F3 antibody (1:100 dilution, Abcam, Cambridge, MA, USA), anti-AXL antibody (1:100 dilution, Abcam, Cambridge, MA, USA) or mouse monoclonal anti-Ki67 antibody (1:100 dilution, Dako, Carpinteria, CA, USA).

Techniques: Mutagenesis, Sequencing, Transfection, Expressing, Western Blot, Control, Software, Luciferase, Activity Assay, Cotransfection

Journal: Oncotarget

Article Title: MicroRNA-432 functions as a tumor suppressor gene through targeting E2F3 and AXL in lung adenocarcinoma

doi: 10.18632/oncotarget.7884

Figure Lengend Snippet: Effects of miR-432 inhibitor transfection on the cisplatin sensitivity were assayed by MTS method in A549 ( A ) and H1299 ( B ). Inhibitor control + Cisplatin vs miR-432 inhibitor + Cisplatin:* p < 0.05; ** p < 0.01. The cell vitality ( C ) and apoptotic fraction ( D ) were analyzed in A549 and H1299 cells after cisplatin treatment at various concentrations with or without miR-432 mimics transfection. Cisplatin + mimics control vs Cispliatin + miR-432 mimics: * p < 0.05; ** p < 0.01. Effects of cisplatin treatment at different concentrations and various time of on miR-432 expression in A549 ( E ) and H1299 ( F ). The expression of E2F3 and AXL mRNA was detected by RT-qPCR in A549 ( G ) and H1299 ( H ) after cisplatin (5 μg/ml) treatment. Western blotting was performed to analyse the expression of AXL (upper) and E2F3 (lower) in A549 ( I ) and H1299 ( J ) as indicated treatments. * p < 0.05.

Article Snippet: The slides were incubated with rabbit polyclonal anti-E2F3 antibody (1:100 dilution, Abcam, Cambridge, MA, USA), anti-AXL antibody (1:100 dilution, Abcam, Cambridge, MA, USA) or mouse monoclonal anti-Ki67 antibody (1:100 dilution, Dako, Carpinteria, CA, USA).

Techniques: Transfection, Control, Expressing, Quantitative RT-PCR, Western Blot

Journal: Oncotarget

Article Title: MicroRNA-432 functions as a tumor suppressor gene through targeting E2F3 and AXL in lung adenocarcinoma

doi: 10.18632/oncotarget.7884

Figure Lengend Snippet: The cell vitality was assayed by MTS after over-expressing E2F3 or AXL in miR-432 mimics transfected A549 ( A ) and H1299 ( B ) cells. E2F3 or AXL expression vector vs empty vector: * p < 0.05. The effects of silencing E2F3 and AXL on miR-432 inhibitor in A549 ( C ) and H1299 ( D ) cells were detected by MTS. siRNA targeting E2F3 or AXL vs NC: * p < 0.05. In the presence of Cisplatin, the effects of over-expressing E2F3 or AXL on the miR-432 mimics transfected A549 ( E ) and H1299 ( F ) cells was detected by MTS. E2F3 or AXL expression vector vs empty vector: * p < 0.05. The same experiments were performed to evaluate the effect of silencing E2F3 or AXL on cisplatin sensitivity in miR-432 inhibitor-transfected A549 ( G ) and H1299 ( H ) cells. siRNA targeting E2F3 or AXL vs NC: * p < 0.05.

Article Snippet: The slides were incubated with rabbit polyclonal anti-E2F3 antibody (1:100 dilution, Abcam, Cambridge, MA, USA), anti-AXL antibody (1:100 dilution, Abcam, Cambridge, MA, USA) or mouse monoclonal anti-Ki67 antibody (1:100 dilution, Dako, Carpinteria, CA, USA).

Techniques: Expressing, Transfection, Plasmid Preparation

Journal: Oncotarget

Article Title: MicroRNA-432 functions as a tumor suppressor gene through targeting E2F3 and AXL in lung adenocarcinoma

doi: 10.18632/oncotarget.7884

Figure Lengend Snippet: ( A ) The percentage of specimens showing low or high miR-32 expression in relation to the expression levels of E2F3 and AXL; * p < 0.05. ( B ) Two representative cases are shown.

Article Snippet: The slides were incubated with rabbit polyclonal anti-E2F3 antibody (1:100 dilution, Abcam, Cambridge, MA, USA), anti-AXL antibody (1:100 dilution, Abcam, Cambridge, MA, USA) or mouse monoclonal anti-Ki67 antibody (1:100 dilution, Dako, Carpinteria, CA, USA).

Techniques: Expressing

Journal: Oncotarget

Article Title: HDAC 1/4-mediated silencing of microRNA-200b promotes chemoresistance in human lung adenocarcinoma cells

doi:

Figure Lengend Snippet: (A) Relative mRNA expression of E2F3, survivin and Aurora-A as measured by qRT-PCR after transfection of sh-control, sh-HDAC1#2 or sh-HDAC4#3 vectors into H1299/DTX and SPC-A1/DTX cells, without (or with) previous transfection of miR-200b inhibitor. Data were normalized by GAPDH and determined relative to sh-control vector group. (B) Protein levels of E2F3, survivin and Aurora-A as measured by western blot after transfection of shRNA-control, shRNA-HDAC1#2 or shRNA-HDAC4#3 vectors into H1299/DTX or SPC-A1/DTX cells, without (or with) previous transfection of miR-200b inhibitor. β-actin was used as an internal control. Data were the means ± standard error of at least three independent experiments. * P < 0.05, ** P < 0.01.

Article Snippet: Hematoxylin and eosin (H&;E) staining, immunostaining of Ki67 (1:500, monoclonal rabbit IgG, tissue culture supernatant), proliferating cell nuclear antigen (PCNA) (1 μg/ml, monoclonal mouse IgG, IgG fraction), HDAC1 (5 μg/ml, monoclonal mouse IgG, protein G purified), HDAC4 (5 μg/ml, polyclonal rabbit IgG, immunogen affinity purified), E2F3 (5 μg/ml, polyclonal rabbit IgG, immunogen affinity purified), Aurora-A (1:500, polyclonal rabbit IgG, immunogen affinity purified) and survivin (1:1000, polyclonal rabbit IgG, protein A purified) (Abcam) staining and TUNEL staining were performed according to the manufacturer's instructions.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Control, Plasmid Preparation, Western Blot, shRNA

Journal: Oncotarget

Article Title: HDAC 1/4-mediated silencing of microRNA-200b promotes chemoresistance in human lung adenocarcinoma cells

doi:

Figure Lengend Snippet: (A) Luciferase activity assays revealed that downregulation of HDAC1/4 significantly elevated Aurora-A (left) or survivin (right) promoter activity partially in a miR-200b/E2F3-dependent manner. Data were normalized by Renilla luciferase activity and determined relative to empty vector group. (B) Downregulation of HDAC1 and HDAC4 decreases the amount of E2F3 binding to the survivin promoter, while suppression of HDAC4 reduces the amount of E2F3 binding to the Aurora-A promoter. ChIP assays were performed with E2F3 antibody in SPC-A1/DTX cells transfected with sh-control and either sh-HDAC1#2 or sh-HDAC4#3. Immunoprecipitated DNA was amplified by qRT-PCR with primers designed to amplify the sequences containing the putative E2F3-binding sites. Data were normalized to qRT-PCR products amplified with input DNA before immunoprecipitation. Data were the means ± standard error of at least three independent experiments. * P < 0.05, ** P < 0.01.

Article Snippet: Hematoxylin and eosin (H&;E) staining, immunostaining of Ki67 (1:500, monoclonal rabbit IgG, tissue culture supernatant), proliferating cell nuclear antigen (PCNA) (1 μg/ml, monoclonal mouse IgG, IgG fraction), HDAC1 (5 μg/ml, monoclonal mouse IgG, protein G purified), HDAC4 (5 μg/ml, polyclonal rabbit IgG, immunogen affinity purified), E2F3 (5 μg/ml, polyclonal rabbit IgG, immunogen affinity purified), Aurora-A (1:500, polyclonal rabbit IgG, immunogen affinity purified) and survivin (1:1000, polyclonal rabbit IgG, protein A purified) (Abcam) staining and TUNEL staining were performed according to the manufacturer's instructions.

Techniques: Luciferase, Activity Assay, Plasmid Preparation, Binding Assay, Transfection, Control, Immunoprecipitation, Amplification, Quantitative RT-PCR

Journal: Oncotarget

Article Title: HDAC 1/4-mediated silencing of microRNA-200b promotes chemoresistance in human lung adenocarcinoma cells

doi:

Figure Lengend Snippet: (A) Growth of tumors in nude mice subcutaneously transplanted with H1299/DTX cells stably transfected with sh-control, sh-HDAC1#2, sh-HDAC4#3 or miR-200b vectors (six mice in each group). Representative photographs of tumors were obtained 6 weeks after inoculation. Data were the means ± standard error. *P < 0.05. (B) The relative mRNA level of miR-200b as measured by qRT-PCR in tumors 6 weeks after inoculation. Data were normalized to U6 RNA and determined relative to control group Data were the means ± standard error. ## P < 0.01 vs. control group. (C) HDAC1, HDAC4, E2F3, survivin and Aurora-A protein levels as measured by western blot in tumors 6 weeks after inoculation. GAPDH was used as an internal control.

Article Snippet: Hematoxylin and eosin (H&;E) staining, immunostaining of Ki67 (1:500, monoclonal rabbit IgG, tissue culture supernatant), proliferating cell nuclear antigen (PCNA) (1 μg/ml, monoclonal mouse IgG, IgG fraction), HDAC1 (5 μg/ml, monoclonal mouse IgG, protein G purified), HDAC4 (5 μg/ml, polyclonal rabbit IgG, immunogen affinity purified), E2F3 (5 μg/ml, polyclonal rabbit IgG, immunogen affinity purified), Aurora-A (1:500, polyclonal rabbit IgG, immunogen affinity purified) and survivin (1:1000, polyclonal rabbit IgG, protein A purified) (Abcam) staining and TUNEL staining were performed according to the manufacturer's instructions.

Techniques: Stable Transfection, Transfection, Control, Quantitative RT-PCR, Western Blot