G9a mediates Sharp-1–dependent inhibition of skeletal muscle differentiation

10.1091/mbc.E12-04-0311Molecular Biology of the Cell23244778-478

G9a mediates Sharp-1–dependent inhibition of skeletal muscle differentiation

10.1091/mbc.E12-04-0311Molecular Biology of the Cell23244778-478

Lysine methyltransferase G9a methylates the transcription factor MyoD and regulates skeletal muscle differentiation

10.1073/pnas.1111628109Proceedings of the National Academy of Sciences1093841-84

Role of Misfolded N-CoR Mediated Transcriptional Deregulation of Flt3 in Acute Monocytic Leukemia (AML)-M5 Subtype

The nuclear receptor co-repressor (N-CoR) is a key component of the generic multi-protein complex involved in transcriptional control. Flt3, a key regulator of hematopoietic cell growth, is frequently deregulated in AML (acute myeloid leukemia). Here, we report that loss of N-CoR-mediated transcriptional control of Flt3 due to misfolding, contributes to malignant growth in AML of the M5 subtype (AML-M5). An analysis of hematopoietic genes in AML cells led to the identification of Flt3 as a transcriptional target of N-CoR. Flt3 level was inversely related to N-CoR status in various leukemia cells. N-CoR was associated with the Flt3 promoter in-vivo, and a reporter driven by the Flt3 promoter was effectively repressed by N-CoR. Blocking N-CoR loss with Genistein; an inhibitor of N-CoR misfolding, significantly down-regulated Flt3 levels regardless of the Flt3 receptor mutational status and promoted the differentiation of AML-M5 cells. While stimulation of the Flt3 receptor with the Flt3 ligand triggered N-CoR loss, Flt3 antibody mediated blockade of Flt3 ligand-receptor binding led to N-CoR stabilization. Genetic ablation of N-CoR potentiated Flt3 ligand induced proliferation of BA/F3 cells. These findings suggest that N-CoR-induced repression of Flt3 might be crucial for limiting the contribution of the Flt3 signaling pathway on the growth potential of leukemic cells and its deregulation due to N-CoR loss in AML-M5, could contribute to malignant growth by conferring a proliferative advantage to the leukemic blasts. Therapeutic restoration of N-CoR function could thus be a useful approach in restricting the contribution of the Flt3 signaling pathway in AML-M5 pathogenesis

A Universal LC-MS/MS Method for Simultaneous Detection of Antibiotic Residues in Animal and Environmental Samples

Detecting and monitoring the usage of antibiotics is a critical aspect of efforts to combat antimicrobial resistance. Antibiotic residue testing with existing LC-MS/MS methods is limited in detection range. Current methods also lack the capacity to detect multiple antibiotic residues in different samples simultaneously. In this study, we demonstrate a methodology that permits simultaneous extraction and detection of antibiotic residues in animal and environmental samples. A total of 30 different antibiotics from 13 classes could be qualitatively detected with our methodology. Further study to reduce analytes’ matrix effect would allow for quantification of antibiotic residues

3D printing biocompatible materials with multi jet fusion for bioreactor applications

In the evolving three-dimensional (3D) printing technology, the involvement of different materials in any new 3D printing process necessitates a thorough evaluation of the product's biocompatibility for biomedical application. Here, we examined the ability of Multi Jet Fusion (MJF)-printed PA-12 to support cell proliferation and osteogenesis. Our results show that leachate from MJF-printed PA-12 does not inhibit the growth of L929 fibroblast and MC3T3e1 osteoblast. The substrate supports the attachment and proliferation of both cell types, though not at a level comparable to conventional polystyrene culture plate. Neither plasma treatment, poly-D-lysine, nor collagen coatings narrowed the gap substantially, suggesting the possible influence of other limiting factors. The substrate can also support MC3T3e1 osteogenesis. However, MJF-printed PA-12 exhibits varying ability in supporting the proliferation of different cell types, especially in subsequent passages. While L929's proliferation is comparable from passage-to-passage, MC3T3e1's growth ability is noticeably compromised. Interestingly, our results show that L929 subcultured back to polystyrene plate retains the ability to grow as robustly as those on the conventional plate, suggesting that MJF-printed PA-12 does not permanently impair cell proliferation. In addition, we have shown the successful culture of bacterial Escherichia coli on MJF-printed PA-12. Together, our study demonstrated the potential of MJF-printed PA-12 for biological applications.Published versionThis research was conducted in collaboration with HP Inc. and supported/partially supported by the Singapore Government through the Industry Alignment Fund-Industry Collaboration Projects Grant

SUMO modification of Stra13 is required for repression of cyclin D1 expression and cellular growth arrest.

Stra13, a basic helix-loop-helix (bHLH) transcription factor is involved in myriad biological functions including cellular growth arrest, differentiation and senescence. However, the mechanisms by which its transcriptional activity and function are regulated remain unclear. In this study, we provide evidence that post-translational modification of Stra13 by Small Ubiquitin-like Modifier (SUMO) dramatically potentiates its ability to transcriptionally repress cyclin D1 and mediate G(1) cell cycle arrest in fibroblast cells. Mutation of SUMO acceptor lysines 159 and 279 located in the C-terminal repression domain has no impact on nuclear localization; however, it abrogates association with the co-repressor histone deacetylase 1 (HDAC1), attenuates repression of cyclin D1, and prevents Stra13-mediated growth suppression. HDAC1, which promotes cellular proliferation and cell cycle progression, antagonizes Stra13 sumoylation-dependent growth arrest. Our results uncover an unidentified regulatory axis between Stra13 and HDAC1 in progression through the G(1)/S phase of the cell cycle, and provide new mechanistic insights into regulation of Stra13-mediated transcriptional repression by sumoylation

Restoration of N-CoR function attenuates the proliferative properties of AML-M5 cells regardless of the Flt3 receptor mutational status.

<p><i>A</i>, Genistein stabilized N-CoR in all 5 AML-M5 cell lines at 50 µM with a concurrent down-regulation of the Flt3 receptor expression as determined via western blotting. <i>B</i>, Restoration of N-CoR function in AML-M5 cells attenuated the proliferative potential of AML-M5 cells. Proliferative capacity of N-CoR positive AML cells HL-60 and N-CoR negative AML-M5 cells after treatment with Genistein at 72 hours was determined via growth proliferation assay (MTT). Growth inhibition was most pronounced at 50 µM in AML-M5 cells while at the same dose, growth inhibitory effects on N-CoR positive HL-60 was less pronounced. Results are representative of 3 independent experiments and asterisks represent p<0.05. <i>C</i>, Morphology of N-CoR positive HL-60 and N-CoR null AML-M5 cells (THP-1, Nomo-1, MV-4-11, SigM5, MM1) as determined via Wright-Giemsa staining after treatment with 50 µM Genistein for 72 hours.</p

Repression of Flt3 by N-CoR protein.

<p><i>A</i>, Relative activity of a luciferase reporter driven by the Flt3 promoter was determined in various leukemic cell lines. The cells were transfected with reporter and reference plasmids using electroporation. The values presented in each bar represent the average of three independent experiments. <i>B</i>, Effect of ectopic N-CoR on the activity of the Flt3 promoter in THP-1 cells electroporated with Flag-tagged N-CoR plasmid in a dose dependent manner was determined via luciferase assay (left panel). The values presented in each bar represent the average of three independent experiments. In parallel, levels of ectopic N-CoR protein in THP-1 cells used in the luciferase assay were determined in western blotting assay with anti-Flag antibody (right panel). <i>C</i>, Effect of ectopic N-CoR on the activity of the Flt3 promoter in 293T cells transfected with N-CoR or control siRNA was determined using luciferase assay. In pGL3-Flt3 (−901) reporter plasmid, luciferase reporter was placed under the control of the full length Flt3 promoter. The values presented in each bar represent the average of three independent experiments. <i>D</i>, The dose dependent fold repression by ectopic N-CoR in N-CoR ablated or non-ablated 293T cells was calculated by dividing the mean relative luciferase activity in N-CoR siRNA transfected cells with that of control siRNA transfected cells of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034501#pone-0034501-g003" target="_blank">Fig. 3C</a>. <i>E</i>, N-CoR is associated with the Flt3 promoter. Relative amounts of Flt3 promoter sequence associated with N-CoR protein in HL-60 or NB4 cells were determined through ChIP assay. The antibody used in the ChIP assay is mentioned at the bottom. N-CoR association with CD36 promoter, a known N-CoR target gene, was determined as positive control.</p