Western blotting (WB), enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FC) have long been used to assess and quantitate relative protein expression in cultured cells and tissue samples. However, WB and ELISA have limited ability to meaningfully quantitate relative protein levels in tissues with complex cell composition, while tissue dissociation followed by FC is not feasible when tissue is limiting and/or cells difficult to isolate. While protein detection in tissue using immunofluorescent (IF) probes has traditionally been considered a qualitative technique, advances in probe stability and confocal imaging allow IF data to be easily quantitated, although reproducible quantitation of relative protein expression requires careful attention to appropriate controls, experiment design, and data collection. Here we describe the methods used to quantify the data presented in Shihan et al. Matrix Biology, 2020 which lays out a workflow where IF data collected on a confocal microscope can be used to quantitate the relative levels of a molecule of interest by measuring mean fluorescent intensity across a region of interest, cell number, and the percentage of cells in a sample “positive” for staining with the fluorescent probe of interest. Overall, this manuscript discusses considerations for collecting quantifiable fluorescent images on a confocal microscope and provides explicit methods for quantitating IF data using FIJI-ImageJ.

EMT and pathogenesis: the good, the bad and the ugly TGF‐b—a central player in EMT Changes in cell–cell/cell–ECM adhesion in EMT—the role of integrins during EMT TGF‐b as a regulator of integrin expression during EMT aV integrins activate TGF‐b ‐ Conformational change activation mechanism ‐ Conformational change activation mechanism with proteolysis TGF‐b and integrin signalling cross‐talk during EMT Disease perspectives Completing the circle Acknowledgements Conflicts of interest Transforming growth factor‐β (TGF‐β) has roles in embryonic development, the prevention of inappropriate inflammation and tumour suppression. However, TGF‐β signalling also regulates pathological epithelial‐to‐mesenchymal transition (EMT), inducing or progressing a number of diseases ranging from inflammatory disorders, to fibrosis and cancer. However, TGF‐β signalling does not proceed linearly but rather induces a complex network of cascades that mutually influence each other and cross‐talk with other pathways to successfully induce EMT. Particularly, there is substantial evidence for cross‐talk between αV integrins and TGF‐β during EMT, and anti‐integrin therapeutics are under development as treatments for TGF‐β‐related disorders. However, TGF‐β’s complex signalling network makes the development of therapeutics to block TGF‐β‐mediated pathology challenging. Moreover, despite our current understanding of integrins and TGF‐β function during EMT, the precise mechanism of their role during physiological versus pathological EMT is not fully understood. This review focuses on the circle of regulation between αV integrin and TGF‐β signalling during TGF‐β induced EMT, which pose as a significant driver to many known TGF‐β‐mediated disorders.

The metastatic potential of cancer cells is directly attributed to their ability to invade through the extracellular matrix. The mechanisms regulating this cellular invasiveness are poorly understood. Here, we show that junctional adhesion molecule A (JAM-A), a tight junction protein, is a key negative regulator of cell migration and invasion. JAM-A is robustly expressed in normal human mammary epithelium, and its expression is down-regulated in metastatic breast cancer tumors. In breast cancer cell lines, an inverse relationship between JAM-A expression and the ability of these cells to migrate on a collagen matrix was observed, which correlates with the known ability of these cells to metastasize. The T47D and MCF-7 cells, which migrate least, are found to express high levels of JAM-A, whereas the more migratory MDA-MB-468 cells have lower levels of JAM-A on the cell surface. MDA-MB-231 cells, which are highly migratory, express the least amount of JAM-A. Overexpression of JAM-A in MDA-MB-231 cells inhibited both migration and invasion through collagen gels. Furthermore, knockdown of JAM-A using short interfering RNAs enhanced the invasiveness of MDA-MB-231 cells as well as T47D cells. The ability of JAM-A to attenuate cell invasion correlated with the formation of increased numbers of focal adhesions and the formation of functional tight junctions. These results show for the first time that an immunoglobulin superfamily cell adhesion protein expressed at tight junctions could serve as a key negative regulator of breast cancer cell invasion and possibly metastasis. Furthermore, loss of JAM-A could be used as a biomarker for aggressive breast cancer. [Cancer Res 2008;68(7):2194–203]

The Pax6 transcription factor functions early during embryogenesis to control key steps in brain, pancreas, olfactory and ocular system development. A requirement for Pax6 in proper formation of lens, iris and retina is well documented. By examining the corneas of heterozygous Small eye (SEY) mice,this report shows that Pax6 is also necessary for normal corneal morphogenesis. In particular, the epithelial component of the postnatal and adult SEY (+/–) cornea is thinner owing to a reduction in the number of cell layers, despite a tenfold increase in the proliferative index and no change in TUNEL labeling. Ultrastructural views revealed large gaps between corneal epithelial cells and a change in the appearance of desmosomes,suggesting that adhesion abnormalities contribute to the corneal phenotype of SEY (+/–) mice. Western blot analysis and immunofluorescence showed equivalent amounts and normal localization of E-cadherin in SEY (+/–)corneas, and the actin cytoskeleton appeared normal as judged by phalloidin staining. By contrast, the levels of desmoglein, β-catenin andγ-catenin were reduced in the SEY (+/–) cornea. In addition, the amount of keratin-12 mRNA and protein, the major intermediate filament, was reduced in SEY (+/–) corneal epithelium as shown by in situ hybridization and immunohistochemistry. Finally, the SEY (+/–) corneal epithelium adheres less well than wild-type when challenged with gentle rubbing using a microsponge. In conclusion, our results indicate that cellular adhesion is compromised in the SEY (+/–) corneal epithelium and suggests a role for Pax6 in the proper generation and maintenance of the adult cornea.