Merging drops in a Teflon tube, and transferring fluid between them, illustrated by protein crystallization and drug screening.

The ability to manipulate drops with small volumes has many practical applications. Current microfluidic devices generally exploit channel geometry and/or active external equipment to control drops. Here we use a Teflon tube attached to a syringe pump and exploit the properties of interfaces between three immiscible liquids to create particular fluidic architectures. We then go on to merge any number of drops (with volumes of micro- to nano-liters) at predefined points in time and space in the tube; for example, 51 drops were merged in a defined order to yield one large drop. Using a different architecture, specified amounts of fluid were transferred between 2 nl drops at specified rates; for example, 2.5 pl aliquots were transferred (at rates of ~500 fl s(-1)) between two drops through inter-connecting nano-channels (width ~40 nm). One proof-of-principle experiment involved screening conditions required to crystallize a protein (using a concentration gradient created using such nano-channels). Another demonstrated biocompatibility; drugs were mixed with human cells grown in suspension or on surfaces, and the treated cells responded like those grown conventionally. Although most experiments were performed manually, moderate high-throughput potential was demonstrated by mixing ~1000 different pairs of 50 nl drops in ~15 min using a robot. We suggest this reusable, low-cost, and versatile methodology could facilitate the introduction of microfluidics into workflows of many experimental laboratories.

Basonuclin-1 modulates epithelial plasticity and TGF-ß1-induced loss of epithelial cell integrity.

Transforming growth factor-ß1 (TGF-ß1) is a multifunctional cytokine and critically involved in the progression of a variety of cancers. TGF-ß1 signaling can impair tumor development by its anti-proliferative and pro-apoptotic features. In contrast, it may actively promote tumor progression and cancer cell dissemination by inducing a gradual switch from epithelial towards mesenchymal-like cell features (EMT-like), including decreased intercellular adhesion. Here, we show that expression of the transcription factor Basonuclin-1 (Bnc1) modulates TGF-ß1-induced epithelial dedifferentiation of mammary epithelial cells. RNAi-mediated repression of Bnc1 resulted in enhanced intercellular adhesion and strongly impaired TGF-ß1-dependent sheet disintegration and cell scattering. In contrast, forced expression of Bnc1 modifies plasma membrane/cytoskeletal dynamics and seemingly interferes with the initiation of sustainable cell-cell contacts. Follow-up analyses revealed that Bnc1 affects the expression of numerous TGF-ß1-responsive genes including distinct EMT-related transcription factors, some of which modulate the expression of Bnc1 themselves. These results suggest that Bnc1 is part of a transcription factor network related to epithelial plasticity with reciprocal feedback-loop connections on which Smad-factors integrate TGF-ß1 signaling. Our study demonstrates that Bnc1 regulates epithelial plasticity of mammary epithelial cells and influences outcome of TGF-ß1 signaling.

The Forkhead factor FoxQ1 influences epithelial differentiation.

The Forkhead family of transcription factors comprises numerous members and is implicated in various cellular functions, including cell growth, apoptosis, migration, and differentiation. In this study, we identified the Forkhead factor FoxQ1 as increased in expression during TGF-ß1 induced changes in epithelial differentiation, suggesting functional roles of FoxQ1 for epithelial plasticity. The repression of FoxQ1 in mammary epithelial cells led to a change in cell morphology characterized by an increase in cell size, pronounced cell-cell contacts, and an increased expression of several junction proteins (e.g., E-cadherin). In addition, FoxQ1 knock-down cells revealed rearrangements in the actin-cytoskeleton and slowed down cell cycle G1-phase progression. Furthermore, repression of FoxQ1 enhanced the migratory capacity of coherent mammary epithelial cells. Gene expression profiling of NM18 cells indicated that FoxQ1 is a relevant downstream mediator of TGF-ß1-induced gene expression changes. This included the differential expression of transcription factors involved in epithelial plasticity, for example, Ets-1, Zeb1, and Zeb2. In summary, this study has elucidated the functional impact of FoxQ1 on epithelial differentiation.