Hedgehog signaling contributes to basic fibroblast growth factor-regulated fibroblast migration.

Fibroblast migration is a central process in skin wound healing, which requires the coordination of several types of growth factors. bFGF, a well-known fibroblast growth factor (FGF), is able to accelerate fibroblast migration; however, the underlying mechanism of bFGF regulation fibroblast migration remains unclear. Through the RNA-seq analysis, we had identified that the hedgehog (Hh) canonical pathway genes including Smoothened (Smo) and Gli1, were regulated by bFGF. Further analysis revealed that activation of the Hh pathway via up-regulation of Smo promoted fibroblast migration, invasion, and skin wound healing, but which significantly reduced by GANT61, a selective antagonist of Gli1/Gli2. Western blot analyses and siRNA transfection assays demonstrated that Smo acted upstream of phosphoinositide 3-kinase (PI3K)-c-Jun N-terminal kinase (JNK)-ß-catenin to promote cell migration. Moreover, RNA-seq and qRT-PCR analyses revealed that Hh pathway genes including Smo and Gli1 were under control of ß-catenin, suggesting that ß-catenin turn feedback activates Hh signaling. Taken together, our analyses identified a new bFGF-regulating mechanism by which Hh signaling regulates human fibroblast migration, and the data presented here opens a new avenue for the wound healing therapy.

A naphthalene derivative as 'turn-on' fluorescent chemosensor for the highly selective and sensitive detection of Al3.

A Schiff base compound derived from naphthalene has been synthesized and characterized as an Al3+ -selective fluorescent probe. The chemosensor (L) exhibits high selectively for Al3+ in aqueous solution, even in the presence of biologically relevant cations such as Na+ , K+ , Ca2+ , Mg2+ , Pb2+ and several transition metal ions. There was no observed interference from anions like Br- , Cl- , HSO3- , SO32- , S2 O32- , NO2- , CO32- and AC- . The lowest detection limit for the chemosensor L was found to be 1.89 × 10-8  M with a linear response towards Al3+ over a concentration range of 5 × 10-6 to 4 × 10-5  M. Furthermore, the proposed chemosensor has been used for imaging of Al3+ in two different types of cells with satisfying results, which further demonstrates its value for practical application in biological systems.

Advanced negative detection method comparable to silver stain for SDS-PAGE separated proteins detection.

In order to achieve an easy, rapid and sensitive protocol to detect proteins in polyacrylamide gel, an advanced negative detection method comparable to silver stain is described. When a gel was incubated with Phloxine B and followed by the development in acidic solution, the zones where forming protein-dye complex were selectively transparent, unlike opaque gel background. Within 50 min after electrophoresis, down to 0.1-0.4 ng of gel-separated proteins (similar with silver stain) could be observed, without labor-intensive and time-consuming procedure. Comparing with the most common negative stain method, Imidazole-zinc stain, Phloxine B stain has been shown higher sensitivity and distinct contrast between the transparent protein bands/spots and opaque background than those; furthermore, it is no longer necessary to concern about retention time of observation. This technique may provide a sensitive and practical choice for proteomics researches.

A rapid and simple 8-quinolinol-based fluorescent stain of phosphoproteins in polyacrylamide gel after electrophoresis.

In order to obtain an easy and rapid protocol to visualize phosphoproteins in SDS-PAGE, a fluorescent detection method named 8-Quinolinol (8-Q) stain is described. 8-Q can form ternary complexes in the gel matrix contributed by the affinity of aluminum ion (Al(3+) ) to the phosphate groups on the proteins and the metal chelating property of 8-Quinolinol, exhibiting strong fluorescence in ultraviolet light. It can visualize as little as 4∼8 ng of α-casein and ß-casein, 16∼32 ng of ovalbumin and κ-casein which is more sensitive than Stains-All but less sensitive than Pro-Q Diamond. The protocol of 8-Q requires only 70 min in 0.75 mm mini-size or 1.0 mm large-size gels with five changes of solutions without destaining step; Pro-Q takes at least 250 min with 11 changes of solutions. In addition, the new method was confirmed by the study of dephosphorylation and LC-MS/MS, respectively. The approach to visualize phosphoprotein utilizing 8-Q could be an alternative to simplify the analytical operations for phosphoproteomics research.

Sequential double fluorescent detections of total proteins and phosphoproteins in SDS-PAGE.

A fluorescent staining technique, using selective chelation with fluorophore and metal ion to the phosphate groups of phosphoproteins in SDS-PAGE is described. As a fluorescent dye and a metal ion, Fura 2 pentapotassium salt and Al(3+) were employed, respectively. The staining method, Fura 2 stain, has sensitivities of 16-32 ng of α-casein and ß-casein, 62 ng of ovalbumin, phosvitin, and κ-casein using an ultraviolet transilluminator. Furthermore, Fura 2 stain is able to carry out continuative double detection of total proteins and phosphoproteins on the same gel within 3.5 h. Consequently, selective phosphoprotein and total protein detections could be obtained without other poststaining. Considering the low cost, simplicity, and speed, Fura 2 staining may provide great practicalities in routine phosphoproteomics research.

Improved conditions for periodate/Schiff's base-based fluorescent staining of glycoproteins with dansylhydrazine in SDS-PAGE.

An improved periodate/Schiff's base based fluorescent stain with dansylhydrazine (DH) for glycoproteins in 1D and 2D SDS-PAGE was described. Down to 4-8 ng of glycoproteins can be selectively detected within 2 h, which is approximately 16-fold higher than that of original protocol, but similar to that of Pro-Q Emerald 488 stain (Invitrogen, Carlsbad, USA). Furthermore, subsequent study of deglycosylation, glycoprotein affinity isolation, and LC-MS/MS analysis were performed to confirm the specificity of the improved method. As a result, improved DH stain may provide a new choice for selective, economic, MS compatible, and convenient visualization of gel-separated glycoproteins.

Alternative visualization of SDS-PAGE separated phosphoproteins by alizarin red S-aluminum (III)-appended complex.

A novel fluorescence detection system using a chemosensor for phosphoprotein in gel electrophoretic analysis has been developed. The system employed the alizarin red S-aluminum (III)-appended complex as a fluorescent staining dye to perform the convenient and selective detection of phosphorylated proteins and total proteins in SDS-PAGE, respectively. Therefore, a full and selective map of proteins can be achieved in the same process without resorting to other compatible detection methods. As low as 62.5 ng of α- (seven or eight phosphates) and ß-casein (five phosphates), 125 ng of ovalbumin (two phosphates), and κ-casein (one phosphate) can be detected in approximately 135 min, with the linear responses of rigorous quantitation of changes over a 125-4000 ng range. As a result, alizarin red S-aluminum (III) stain may provide a new choice for selective, economic, and convenient visualization of phosphoproteins.

Fluorescent staining of protein in sodium dodecyl sulfate polyacrylamide gels by salicylaldehyde azine.

As a non-covalent fluorescence probe, in this study, salicylaldehyde azine (SA) was introduced as a sensitive fluorescence-based dye for detecting proteins both in 1-D and 2-D polyacrylamide electrophoresis gels. Down to 0.2 ng of single protein band could be detected within 1 h, which similars to that of glutaraldehyde (GA)-silver stain, but approximately four times higher than that of SYPRO Ruby fluorescent stain. Furthermore, comparative analysis of the MS compatibility of SA stain with SYPRO Ruby stain indicated that SA stain is compatible with the downstream of protein identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Additionally, the probable mechanism of the SA stain was investigated by molecular docking. The results demonstrated that the interaction between SA and protein was mainly contributed by hydrogen bonding and hydrophobic forces.

Improved staining of phosphoproteins with high sensitivity in polyacrylamide gels using Stains-All.

An improved Stains-All (ISA) staining method for phosphoproteins in SDS-PAGE was described. Down to 0.5-1 ng phosphoproteins (α-casein, ß-casein, or phosvitin) can be successfully selectively detected by ISA stain, which is approximately 120-fold higher than that of original Stains-All stain, but is similar to that of commonly used Pro-Q Diamond stain. Furthermore, unlike the original Stains-All protocol that was time consuming and light unstable, ISA stain could be completed within 60 min without resorting to protect the gels from light during the whole staining procedure. According to the results, it is concluded that ISA stain is a rapid, sensitive, specific, and economic staining method for a broad application to the research of phosphoproteins.

Phosphoprotein staining for sodium dodecyl sulfate-polyacrylamide gel electrophoresis using fluorescent reagent morin hydrate.

A fluorescence-based stain with 3,5,7,2',4'-pentahydroxyflavone (morin hydrate, MH) was designed to stain phosphoproteins in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Al(3+) was applied as a "fixed bridge," providing an efficient energy transfer channel between phosphoprotein and MH, to produce a strong fluorescent complex for the determination of phosphoprotein. As little as 62.5ng of α-casein (7 or 8 phosphates) and ß-casein (5 phosphates), 125ng of ovalbumin (2 phosphates), and κ-casein (1 phosphate) could be visualized with a wide linear dynamic range. In comparison with conventional methods, MH stain is a time-saving method that takes just 90min. It also has good compatibility with routine protein stainings such as Coomassie Brilliant Blue R (CBBR) and SYPRO Ruby for total protein analysis.

Proteomic study on the protective mechanism of fibroblast growth factor 21 to ischemia-reperfusion injury.

Fibroblast growth factor (FGF)-21 is a novel regulator of insulin-independent glucose transport in 3T3-L1 adipocytes and has glucose and triglyceride lowering effects in rodent models of diabetes. In this study, we found that FGF-21 can significantly attenuate ischemia-reperfusion (I/R) induced damage in H9c2 cells (rat heart). However, it is unclear which signal transduction pathway is involved in the cardioprotective effect of FGF-21. Thus, this study was designed to investigate the potential mechanism induced by FGF-21. The results showed that FGF-21 treatment prevented the oxidative stress and apoptosis associated with I/R damage by reducing the levels of superoxide anions, inhibiting glycogen synthase kinase (GSK) 3ß by activating Akt phosphorylation, and recovering the levels of ATP synthase pyruvate kinase isozymes M1 and protein kinase C, thereby improving energy supply. In summary, we conclude that FGF-21 protects H9c2 cells against I/R injury mainly through the Akt-GSK-3ß-caspase-3 dependent pathway, preventing oxidative stress, and recovery of the energy supply.

An improved silver stain for the visualization of lipopolysaccharides on polyacrylamide gels.

A sensitive, brief, and user-friendly silver stain to meet the needs in high-efficiency detection of lipopolysaccharides (LPS) on polyacrylamide gels is described. In this study, the most commonly used formaldehyde-based LPS silver stain, which is potentially hazardous to the operator, is replaced by ascorbic acid (Vc) in alkaline sodium thiosulfate solution. It takes only about 35 min to complete all the protocol, with a detection limit of 4 ng of total LPS. The results indicate that this user-friendly method could be a good choice for LPS visualization on polyacrylamide gels.

Negative staining of lipopolysaccharides on polyacrylamide gels by using eosin B.

A sensitive and simple technique for the negative detection of lipopolysaccharides (LPSs) following polyacrylamide gel electrophoresis (PAGE) using eosin B (EB) was developed. After electrophoresis, gels were fixed, stained, and developed within 30 min to achieve transparent and colorless LPS bands under opaque gel matrix background. As low as 20 to 40 ng of total LPSs could be detected, which is 4-fold more sensitive than those of the widely used silver stain developed by Fomsgaard and coworkers and imidazole-zinc (IZ) negative stain. For its sensitivity and brevity, this stain may be a practical method for LPS determination in the routine laboratory.

GSK-3 phosphorylates delta-catenin and negatively regulates its stability via ubiquitination/proteosome-mediated proteolysis.

Delta-catenin was first identified because of its interaction with presenilin-1, and its aberrant expression has been reported in various human tumors and in patients with Cri-du-Chat syndrome, a form of mental retardation. However, the mechanism whereby delta-catenin is regulated in cells has not been fully elucidated. We investigated the possibility that glycogen-synthase kinase-3 (GSK-3) phosphorylates delta-catenin and thus affects its stability. Initially, we found that the level of delta-catenin was greater and the half-life of delta-catenin was longer in GSK-3beta(-/-) fibroblasts than those in GSK-3beta(+/+) fibroblasts. Furthermore, four different approaches designed to specifically inhibit GSK-3 activity, i.e. GSK-3-specific chemical inhibitors, Wnt-3a conditioned media, small interfering RNAs, and GSK-3alpha and -3beta kinase dead constructs, consistently showed that the levels of endogenous delta-catenin in CWR22Rv-1 prostate carcinoma cells and primary cortical neurons were increased by inhibiting GSK-3 activity. In addition, it was found that both GSK-3alpha and -3beta interact with and phosphorylate delta-catenin. The phosphorylation of DeltaC207-delta-catenin (lacking 207 C-terminal residues) and T1078A delta-catenin by GSK-3 was noticeably reduced compared with that of wild type delta-catenin, and the data from liquid chromatography-tandem mass spectrometry analyses suggest that the Thr(1078) residue of delta-catenin is one of the GSK-3 phosphorylation sites. Treatment with MG132 or ALLN, specific inhibitors of proteosome-dependent proteolysis, increased delta-catenin levels and caused an accumulation of ubiquitinated delta-catenin. It was also found that GSK-3 triggers the ubiquitination of delta-catenin. These results suggest that GSK-3 interacts with and phosphorylates delta-catenin and thereby negatively affects its stability by enabling its ubiquitination/proteosome-mediated proteolysis.

Promoted negative staining of proteins in SDS-PAGE using Eosin B compounded with magnesium chloride.

In this study, we describe an effective visualizing technique for proteins in SDS-PAGE based on the organic dye, Eosin B, the sensitivity of which can be further strengthened by the addition of magnesium to the staining solution after electrophoresis. The newly developed protocol is low in cost and easily performed compared with the common methods for protein analysis in 1-D and 2-D gels. It provides a much better sensitivity (0.2 ng of single protein band) than that of imidazole-zinc negative stain for fixing and staining within 1 h, and an excellent performance in terms of compatibility with MALDI-TOF MS. The results show that similar identification scores and numbers of matched peptides were obtained by both methods. Furthermore, the effects of different metal salts on the quality of protein visualization by Eosin B were also investigated. Because of its sensitivity, stability, and safety, this stain may be a more practical method for protein determination in the routine laboratory.

High-throughput negative detection of SDS-PAGE separated proteins and its application for proteomics.

A negative detection method for proteins on SDS-PAGE is described. In this method, Eosin Y (EY) was selectively precipitated in the gel background, which is absent from those zones where proteins are located through the formation of a stable water-soluble protein-dye complex. Negative staining of proteins using EY, allows high-sensitivity, low-cost, and simple protocol. The new described method takes less than an hour to complete all the protocol, with a detection limit of 0.5 ng of single protein band. Comparing with imidazole-zinc negative stain, EY dye provides broader linear dynamic range, higher sensitivity and reproducibility, and better obvious contrast between the protein bands or spots and background. Furthermore, the novel technique developed here presented a real practical method for simultaneous processing of multiple gels, which makes it possible to perform high-throughput staining for proteome research. Additionally, we have also compared the influence of staining method on the quality of mass spectra by PMF.

A user-friendly alternative to formaldehyde-based DNA silver-staining method on polyacrylamide gels.

We have developed a practical, cost-effective and user-friendly protocol to meet the needs of nucleic acids research, particularly in respect of DNA detection on polyacrylamide gels. In this method, the most commonly used alkaline formaldehyde developer in DNA silver stain, which does harm to operator, is first replaced by glucose in alkaline borate buffer. In addition, the effects of six reducing sugars on the quality of DNA visualization were investigated. Consequently, the optimal protocol using glucose takes about 45 min to complete all the procedures, with a detection limit of 5 pg of single DNA band on polyacrylamide gels, was developed. The results indicate that this user-friendly and economic protocol could be a good choice for routine use in DNA visualization on polyacrylamide gels.

Improved conditions for silver-ammonia staining of DNA in polyacrylamide gel.

An improved silver-ammonia staining method for DNA on polyacrylamide gels is described. In this method, staining of DNA using silver-ammonia complex allows high sensitivity, low cost, low toxicity, and simple protocol without requiring fixation and sensitization steps. The protocol takes less than 40 min to complete, with a detection limit of 1.5 pg of single DNA band on polyacrylamide gels, approximately 30-fold higher than that of original silver-ammonia staining method. Furthermore, this novel technique not only exhibits high sensitivity for large DNA fragment, but also shows a better trend to detect low-base-pair DNA compared with other silver staining methods.

An expanding negative detection method for the visualization of DNA in polyacrylamide gels by eosin Y.

A sensitive and easy technique has been developed for the negative detection of DNA following PAGE using eosin Y. After electrophoresis, gels are fixed and stained within 40 min to provide a detection limit of 0.1-0.2 ng of single DNA band, which appears as transparent and colorless under the opaque gel matrix background. The sensitivity of the new stain is fourfold better than zinc-imidazole negative and ethidium bromide stains. Furthermore, the newly developed staining method has been successfully applied to RNA visualization in polyacrylamide gels. In addition, the inclusion of inorganic salts in staining solution was also investigated, which has great effect on the staining efficiency.

A visible dye-based staining method for DNA in polyacrylamide gels by ethyl violet.

We describe a visible dye-based staining method for DNA in polyacrylamide gels using ethyl violet (EV). The novel method is a background-free, sensitive, economical, and simple procedure involving only staining and washing steps that can be completed within 30 min. As little as 0.8-1.6 ng of phiX174 DNA/HaeIII can be detected by EV, which is about eightfold more sensitive than Nile blue (NB) stain and twofold less sensitive than ethidium bromide (EB) stain.