Myofibroblast Markers and Microscopy Detection Methods in Cell Culture and Histology.

The identification of myofibroblasts is essential for mechanistic in vitro studies, cell-based drug tests, and to assess the level of fibrosis in experimental animal or human fibrosis. The name myo-fibroblast was chosen in 1971 to express that the formation of contractile features-stress fibers is the essential criterion to define these cells. Additional neo-expression of α-smooth muscle actin (α-SMA) in stress fibers has become the most widely used molecular marker. Here, we briefly introduce the concept of different myofibroblast activation states, of which the highly contractile α-SMA-positive phenotype represents a most advanced functional stage. We provide targeted immunofluorescence protocols to assess this phenotype, and publicly accessible image analysis tools to quantify the level of myofibroblast activation in culture and in tissues.

ARID1A facilitates KRAS signaling-regulated enhancer activity in an AP1-dependent manner in colorectal cancer cells.

BACKGROUND: ARID1A (AT-rich interactive domain-containing protein 1A) is a subunit of the BAF chromatin remodeling complex and plays roles in transcriptional regulation and DNA damage response. Mutations in ARID1A that lead to inactivation or loss of expression are frequent and widespread across many cancer types including colorectal cancer (CRC). A tumor suppressor role of ARID1A has been established in a number of tumor types including CRC where the genetic inactivation of Arid1a alone led to the formation of invasive colorectal adenocarcinomas in mice. Mechanistically, ARID1A has been described to largely function through the regulation of enhancer activity. METHODS: To mimic ARID1A-deficient colorectal cancer, we used CRISPR/Cas9-mediated gene editing to inactivate the ARID1A gene in established colorectal cancer cell lines. We integrated gene expression analyses with genome-wide ARID1A occupancy and epigenomic mapping data to decipher ARID1A-dependent transcriptional regulatory mechanisms. RESULTS: Interestingly, we found that CRC cell lines harboring KRAS mutations are critically dependent on ARID1A function. In the absence of ARID1A, proliferation of these cell lines is severely impaired, suggesting an essential role for ARID1A in this context. Mechanistically, we showed that ARID1A acts as a co-factor at enhancers occupied by AP1 transcription factors acting downstream of the MEK/ERK pathway. Consistently, loss of ARID1A led to a disruption of KRAS/AP1-dependent enhancer activity, accompanied by a downregulation of expression of the associated target genes. CONCLUSIONS: We identify a previously unknown context-dependent tumor-supporting function of ARID1A in CRC downstream of KRAS signaling. Upon the loss of ARID1A in KRAS-mutated cells, enhancers that are co-occupied by ARID1A and the AP1 transcription factors become inactive, thereby leading to decreased target gene expression. Thus, targeting of the BAF complex in KRAS-mutated CRC may offer a unique, previously unknown, context-dependent therapeutic option in CRC.

Ig-like Domain in Endoglucanase Cel9A from Alicyclobacillus acidocaldarius Makes Dependent the Enzyme Stability on Calcium.

Endoglucanase Cel9A from Alicyclobacillus acidocaldarius (AaCel9A) has an Ig-like domain and the enzyme stability is dependent to calcium. In this study the effect of calcium on the structure and stability of the wild-type enzyme and the truncated form (the wild-type enzyme without Ig-like domain, AaCel9AΔN) was investigated. Fluorescence quenching results indicated that calcium increased and decreased the rigidity of the wild-type and truncated enzymes, respectively. RMSF results indicated that AaCel9A has two flexible regions (regions A and B) and deleting the Ig-like domain increased the truncated enzyme stability by decreasing the flexibility of region B probably through increasing the hydrogen bonds. Calcium contact map analysis showed that deleting the Ig-like domain decreased the calcium contacting residues and their calcium binding affinities, especially, in region B which has a role in calcium binding site in AaCel9A. Metal depletion and activity recovering as well as stability results showed that the structure and stability of the wild-type and truncated enzymes are completely dependent on and independent of calcium, respectively. Finally, one can conclude that the deletion of Ig-like domain makes AaCel9AΔN independent of calcium via decreasing the flexibility of region B through increasing the hydrogen bonds. This suggests a new role for the Ig-like domain which makes AaCel9A structure dependent on calcium.

Deleting the Ig-Like Domain of Alicyclobacillus acidocaldarius Endoglucanase Cel9A Causes a Simultaneous Increase in the Activity and Stability.

Endoglucanase Cel9A from Alicyclobacillus acidocaldarius (AaCel9A) is a monomeric enzyme with 537 residues. This enzyme has an Ig-like domain in the N-terminus of the catalytic domain. In this study, the role of the Ig-like domain on the activity, stability, and structural rigidity of AaCel9A and the effect of calcium on enzyme activity and stability were examined by comparing a truncated enzyme with deletion of the Ig-like domain (AaCel9AΔN) to the wild-type enzyme. Our results showed that the deletion of the Ig-like domain increased the catalytic efficiency of the truncated enzyme up to threefold without any significant changes in the K m of the enzyme. Furthermore, pH and temperature optimum for activity were shifted from 6.5 to 7.5 and from 65 to 60 °C, respectively, by deletion of the Ig-like domain. The thermal stability and fluorescence quenching results indicated that the stability and rigidity of the truncated enzyme have been more than that of the wild-type enzyme. Calcium similarly increased the catalytic efficiency of the enzymes (up to 40 %) and remarkably raised the stability of the AaCel9A compared to the AaCel9AΔN. This shows that Ig-like domain has a role in the increase of the enzyme stability by calcium in the wild-type enzyme.