The possible roles of Src family kinases in the enhanced malignant properties of melanomas related to GD3 expression were analyzed. Among Src family kinases only Yes, not Fyn or Src, was functionally involved in the increased cell proliferation and invasion of GD3-expressing transfectant cells (GD3؉). Yes was located upstream of p130Cas and paxillin and at an equivalent level to focal adhesion kinase. Yes underwent autophosphorylation even before serum treatment and showed stronger kinase activity in GD3؉ cells than in GD3؊ cells following serum treatment. Coimmunoprecipitation experiments revealed that Yes bound to focal adhesion kinase or p130Cas more strongly in GD3؉ cells than in GD3؊ cells. As a possible mechanism for the enhancing effects of GD3 on cellular phenotypes, it was shown that majority of Yes was localized in glycolipidenriched microdomain/rafts in GD3؉ cells even before serum treatment, whereas it was scarcely detected in glycolipid-enriched microdomain/rafts in GD3؊ cells. An in vitro kinase assay of Yes revealed that coexistence of GD3 with Yes in membranous environments enhances the kinase activity of GD3؊ cell-derived Yes toward enolase, p125, and Yes itself. Knockdown of GD3 synthase resulted in the alleviation of tumor phenotypes and reduced activation levels of Yes. Taken together, these results suggest a role of GD3 in the regulation of Src family kinases.

Epithelial–mesenchymal transition (EMT) is a cellular process involved in development and disease progression. Intermediate EMT states were observed in tumors and fibrotic tissues, but previous in vitro studies focused on time-dependent responses with single doses of signals; it was unclear whether single-cell transcriptomes support stable intermediates observed in diseases. Here, we performed single-cell RNA-sequencing with human mammary epithelial cells treated with multiple doses of TGF-β. We found that dose-dependent EMT harbors multiple intermediate states at nearly steady state. Comparisons of dose- and time-dependent EMT transcriptomes revealed that the dose-dependent data enable higher sensitivity to detect genes associated with EMT. We identified cell clusters unique to time-dependent EMT, reflecting cells en route to stable states. Combining dose- and time-dependent cell clusters gave rise to accurate prognosis for cancer patients. Our transcriptomic data and analyses uncover a stable EMT continuum at the single-cell resolution, and complementary information of two types of single-cell experiments.

HighlightsWe developed a quality assessment method applicable to single-cell RNA-Seq data SkewC relies on the use of gene coverage profile of cells as a quality measure SkewC reveals two classes of cells: typical cells and skewed cells Typical with prototypical coverage profiles, and skewed with skewed profiles

Epithelial-mesenchymal transition (EMT) is a key cellular process involved in development and disease progression. Single-cell transcriptomes can characterize intermediate EMT states observed in tumors and fibrotic tissues, but previous in vitro models focused on time-dependent responses after stimulation with single dose of EMT signals. It was therefore unclear whether single-cell transcriptomes support stable intermediate EMT phenotypes crucial for disease progression. We performed single-cell RNA-sequencing with human mammary epithelial cells treated with various concentrations of TGF-β. We found that the dose-dependent EMT harbors multiple intermediate states at the single-cell level after two weeks of treatment, suggesting a stable continuum. After correcting batch effects from experiments, we performed comparative analyses of the dose- and time-dependent EMT. We found that the dose-dependent EMT shows a stronger anti-correlation between epithelial and mesenchymal transcriptional programs and a better resolution of transition stages compared to the time-dependent process. These properties enable higher sensitivity to detect genes whose expressions are associated with core EMT regulatory networks. Nonetheless, we found cell clusters unique to the time-dependent EMT, which correspond to en route cell populations that do not appear at steady states. Furthermore, combining dose- and time-dependent cell clusters gave rise to more accurate prognosis for cancer patients compared to individual EMT spectrum. Our new data and analyses reveal a stable EMT continuum at the single-cell resolution and the transcriptomic level. The dose-dependent experimental model can complement the widely used time-course experiments to reflect physiologically or pathologically relevant EMT phenotypes in a comprehensive manner.

Activation of the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-jB) transcription factor, a central player in immune response regulation, is based on phosphorylation of inhibitor of kappaB alpha (IjBa) by the Inhibitor of kappaB kinase (IKK) that triggers IjBa degradation. Although inhibitor of kappaB beta (IjBb) is structurally similar to IjBa, its precise characteristics remain undefined. Herein, we report that the molecular interactivity of IjBb with the kinase-active region of IKK subunit 2 (IKK2), as well as its phosphorylation status, differs markedly from those of IjBa. A mass spectrometry analysis revealed that IjBb phosphorylation sites are distributed in its C-terminal region, whereas IjBa phosphorylation sites are located in the N-terminal region. Furthermore, IKK2 phosphorylation sites in IjBb are found in a region distinct from typical degradation signals, such as phosphodegron and proline/glutamic acid/serine/threonine-rich sequence (PEST) motifs. Mutation of the IjBb phosphorylation sites enhances its resistance to homeostatic proteasomal degradation. These findings contribute a novel concept in NF-jB/IKK signalling research.