A powerful method to study gene function is expression or overexpression in an inducible, cell type-specific system followed by observation of consequent phenotypic changes and visualization of linked reporters in the target tissue. Multiple inducible gene overexpression systems have been developed for plants, but very few of these combine plant selection markers, control of expression domains, access to multiple promoters and protein fusion reporters, chemical induction, and high-throughput cloning capabilities. Here, we introduce a MultiSite Gateway-compatible inducible system for Arabidopsis (Arabidopsis thaliana) plants that provides the capability to generate such constructs in a single cloning step. The system is based on the tightly controlled, estrogen-inducible XVE system. We demonstrate that the transformants generated with this system exhibit the expected cell type-specific expression, similar to what is observed with constitutively expressed native promoters. With this new system, cloning of inducible constructs is no longer limited to a few special cases but can be used as a standard approach when gene function is studied. In addition, we present a set of entry clones consisting of histochemical and fluorescent reporter variants designed for gene and promoter expression studies.

Pemphigus is an autoimmune cutaneous disease characterized by circulating autoantibodies that cause blistering and erosions on skin and mucous membranes. Circulating autoantibodies bind to epidermal cell membrane and cause cell-cell detachment (acantholysis), leading to epidermal tissue damage and cell death. The principal target of pemphigus vulgaris autoantibodies (PV-IgG) is desmosomal cadherin desmoglein 3 (Dsg3), a constituent of desmosomes, mediating cell-cell adhesion. Several hypotheses for the mechanisms of acantholysis induction by PV-IgG exist, but the actual mechanism is not clear as yet. We have previously reported on apoptosis induction in PV-IgG-mediated epidermal tissue and cell damage as a possible mechanism of acantholysis and cell death (Wang et al. 2004, Apoptosis, 9:131-143). In this study we investigated the involvement of the EGFR and intracellular signal transduction pathways in the PV-IgG-induced apoptosis. We show here that PV-IgG induced activation/autophosphorylation of EGFR in cultured keratinocytes in vitro. The specific tyrosine kinase inhibitor AG1478 abrogated EGFR autophosphorylation, cell death, FasL appearance and acantholysis, all induced by PV-IgG, in parallel, confirming the involvement of EGFR in this Fas apoptotic cascade. Activation of EGFR was followed by phosphorylation of its downstream substrates, MAP kinase ERK and transcription factor c-Jun, and internalization of EGFR. Pharmacological inactivation of the EGFR and ERK kinase activities, by use of specific inhibitors AG1478 and PD98059 respectively, blocked PV-IgG-induced phosphorylation of EGFR, ERK and c-Jun and cellular apoptosis, measured by flow cytometry and caspase 3 activity. Prolonged activation of EGFR by PV-IgG led to dramatic internalization of this receptor, possibly reducing the ability of the cell to perform survival signals. This suggests that activation of EGFR, followed by its internalization, is pivotal for intracellular apoptotic signal transduction via ERK/c-Jun pathways, leading to acantholysis. Our experimental data indicate that the EGFR is instrumental in transducing apoptotic/acantholytic signals in keratinocytes cultures in response to PV-IgG treatment. The acantholytic effect caused by PV-IgG binding to cell surface receptors begins with and depends on cell surface receptor (EGFR) activation of intracellular signaling pathways (ERK pathway) and apoptosis induction (FasR pathway), which later lead to major cell-cell separation (acantholysis) and cell death.

Silkworms (Bombyx mori) produce massive amounts of silk proteins to make cocoons during the final stages of larval development. Although the major components, fibroin and sericin, have been the focus for a long time, few researchers have realized the complexity of the silk proteome. We collected seven kinds of silk fibers spun by silkworm larvae at different developmental stages: the silks spun by new hatched larvae, second instar day 0 larvae, third instar day 0 larvae, fourth instar day 0 larvae, and fourth instar molting larvae, the scaffold silk used to attach the cocoon to the substrate and the cocoon silk. Analysis by liquid chromatography-tandem mass spectrometry identified 500 proteins from the seven silks. In addition to the expected fibroins, sericins, and some known protease inhibitors, we also identified further protease inhibitors, enzymes, proteins of unknown function, and other proteins. Unsurprisingly, our quantitative results showed fibroins and sericins were the most abundant proteins in all seven silks. Except for fibroins and sericins, protease inhibitors, enzymes, and proteins of unknown function were more abundant than other proteins. We found significant change in silk protein compositions through development, being consistent with their different biological functions and complicated formation.

We investigated the role of actin filament associated protein 1 antisense RNA1 (AFAP1-AS1) lncRNA in promoting cholangiocarcinoma (CCA). qRT-PCR analysis of patient samples showed that AFAP1-AS1 expression was higher in CCA tumors than matched adjacent non-tumor tissue. AFAP1-AS1 levels were also higher in CCA cell lines (HuCCT1 and TFK-1) than a normal biliary epithelium cell line (HIBEpic). AFAP1-AS1 knockdown in CCA cell lines using shAFAP1-AS1 reduced cell proliferation and colony formation in CCK-8 and colony formation assays, respectively. Cell cycle analysis demonstrated that AFAP1-AS1 knockdown resulted in G0/G1 cell cycle arrest and inhibition of S-G2/M transition compared to the controls. CCA cells transfected with shAFAP1-AS1 also exhibited reduced metastasis and invasiveness in Transwell and wound healing assays. This was further confirmed in xenograft experiments with nude mice using CCA cells transfected with shAFAP1-AS1 or control shRNA. AFAP1-AS1 knockdown cells produced smaller tumors, demonstrating that AFAP1-AS1 promotes tumor growth in vivo. AFAP1-AS1 knockdown also increased expression of actin filament associated protein 1 (AFAP1) and reduced cell stress filament integrity, as determined from western blot and immunofluorescence assays, respectively. These findings indicate that AFAP1-AS1 exerts oncogenic effects in CCA. We postulate that AFAP1-AS1 is a potentially useful diagnostic and prognostic biomarker and therapeutic target for CCA.

Clinical trials have demonstrated the importance of aromatase inhibitor (AI) therapy in the effective treatment of hormone-dependent breast cancers. Yet, as with all prolonged drug therapy, resistance to aromatase inhibitors does develop. To date, the precise mechanism responsible for resistance to aromatase inhibitors is not completely understood. In this paper, several mechanisms of de novo/ intrinsic resistance and acquired resistance to AIs are discussed. These mechanisms are hypothesized based on important findings from a number of laboratories.To better understand this question, our lab has generated, in vitro, breast cancer cell lines that are resistant to aromatase inhibitors. Resistant cell lines were generated over a prolonged period of time using the MCF-7aro (aromatase overexpressed) breast cancer line. These cell lines are resistant to the aromatase inhibitors letrozole, anastrozole and exemestane and the anti-estrogen tamoxifen, for comparison. Two types of resistant cell lines have been generated, those that grow in the presence of Testosterone (T) which is needed for cell growth, and resistant lines that are cultured in the presence of inhibitor only (no T). In addition to functional characterization of aromatase and ERα in these resistant cell lines, microarray analysis has been employed in order to determine differential gene expression within the aromatase inhibitor resistant cell lines versus tamoxifen, in order to better understand the mechanism responsible for AI resistance on a genome-wide scale. We anticipate that our studies will generate important information on the mechanisms of AI resistance. Such information can be valuable for the development of treatment strategies against AI resistant breast cancers.