Journal: Oncotarget

Article Title: Estrogen Receptor Alpha Prevents Bladder Cancer Development via INPP4B inhibited Akt Pathway in vitro and in vivo

doi:

Figure Lengend Snippet: (A) ERα expression was detected by antibody (middle 2 panels) and DAPI immunofluorescence staining was used to detect nuclei (left 2 panels) in SVHUC/ERα+ and SVHUC/vector cells. Non-malignant SVHUC cells, with or without ERα expression, were subjected to malignant transformation by MCA carcinogen treatment for three cycles and cultured for another six weeks. Soft agar assays (right panels) were performed to analyze cell transformation ability. Quantitative analyses (far right panel) of the colony numbers. ***, p<0.001 by unpaired t -test. (B) MTT assay used to analyze cell growth in 647v (shGFP or shERα), (C) UMUC3 (vector or ERα), and (D) T24 (vector or ERα) cells. ***, p<0.001 by Two-way ANOVA test. (E) Anchorage independent cell growth assay of BCa cells, infected with lentiviral shRNA against GFP or ERα in 647v cells, and lentoviral ERα or vector control in UMUC3 cells. *, p<0.05, ***, p<0.001 compared to control cells by t-test. (F) Knockdown of ERα increases tumor growth in the 647v xenograft mouse model. Nude mice were injected with 647v cancer cells expressing shRNA against ERα or control GFP in both flanks. Mice were sacrificed six weeks after cancer cell implantations. Mice injected with 647v cells with ERα shRNA showed increased tumor weights compared to shGFP control group (left panel). Quantification of tumor weights at right (p<0.01 by t -test).

Article Snippet: The short hairpin RNA (shRNA) expressing lentiviral vector against GFP (PLKO.1-puro-shGFP) and PLKO.1 hygro vector were obtained from Addgene.

Techniques: Expressing, Immunofluorescence, Staining, Plasmid Preparation, Transformation Assay, Cell Culture, MTT Assay, Growth Assay, Infection, shRNA, Control, Knockdown, Injection

Journal: Oncotarget

Article Title: Estrogen Receptor Alpha Prevents Bladder Cancer Development via INPP4B inhibited Akt Pathway in vitro and in vivo

doi:

Figure Lengend Snippet: UMUC3 (A) and T24 (B) were transduced with lentiviral ERα and/or shINPP4B to investigate the functional connection of ERα and INPP4B in BCa cell growth. BCa cells were first infected with lentiviral ERα or vector (ERα(+), vec) and then further infected with lentiviral shINPP4B or sh control. We compared 3 groups of cells: (i) vector/shCon, (ii) ERα+/shCon, or (iii) ERα+/shINPP4B to assay cell growth on Days 0, 2, 4 and 6. ***, p<0.001 by Two-way ANOVA test. (C) SVHUC cells were transduced with lentiviral ERα and/or shINPP4b to investigate the functional connection of ERα and INPP4B in bladder cell malignant transformation. SVHUC cells with vector/shCon, ERα(+)/shCon, or ERα(+)/shINPP4B transduction were subjected to MCA carcinogen treatment. Soft agar assays were performed to analyze malignant transformation ability. Quantitative analysis of the colony numbers of the soft agar assays is shown. ***, p<0.001 by one way ANOVA test. (D) Protein lysates were collected from SVHUC, UMUC3, and T24 cells with lentiviral vector/shCon, ERα(+)/shCon, and ERα(+)/shINPP4B transduction. Immunoblotting was performed with antibodies against ERα, INPP4B, and GAPDH. (E) Protein lysates were collected from UMUC3 cells with lentiviral vector/shCon, ERα(+)/shCon, and ERα(+)/shINPP4B transduction. Immunoblotting was performed with antibodies against ERα, INPP4B, phosphorylated AKT (pAKT), total AKT (tAKT), and GAPDH.

Article Snippet: The short hairpin RNA (shRNA) expressing lentiviral vector against GFP (PLKO.1-puro-shGFP) and PLKO.1 hygro vector were obtained from Addgene.

Techniques: Transduction, Functional Assay, Infection, Plasmid Preparation, Control, Transformation Assay, Western Blot