Targeted Quantitative Proteomics Revealed Arsenite-induced Proteasomal Degradation of RhoB in Fibroblast Cells

Arsenic is a toxicant widely present in the environment. Previous epidemiological and animal studies support that arsenic exposure is associated with elevated incidences of lung and skin cancers. Therefore, it is important to understand the molecular mechanisms through which arsenite initiates malignant transformation of lung and skin tissues. Ras superfamily of small GTPases assumes a crucial role in many cellular processes including transcription, protein synthesis, and trafficking. In addition, small GTPase signaling is known to be altered in many types of cancer. By employing a multiple-reaction monitoring (MRM)-based targeted proteomic method, we found that the protein level of RhoB was substantially decreased in IMR90 human lung fibroblast cells upon a 12-h exposure to 5 μM NaAsO2. In addition, the protein level of ectopically expressed RhoB was found to decline in a dose-dependent manner upon arsenite exposure in HEK293T, HeLa, and GM00637 cells as well as that of endogenous RhoB protein in IMR90 cells. Moreover, the arsenite-elicited down-regulation of RhoB was found to arise from enhanced proteasomal degradation. Taken together, we demonstrated, for the first time, that exposure to arsenite could attenuate the protein expression of RhoB through proteasomal degradation

Data for: Comparative metabolite profiling of a metastatic and primary melanoma cell line using untargeted metabolomics: a case study

These data represent the core tandem mass spectra and qPCR reports supporting the manuscript: Comparative metabolite profiling of a metastatic and primary melanoma cell line using untargeted metabolomics: a case stud

Long non-coding RNA KCNQ1OT1 overexpression promotes osteogenic differentiation of staphylococcus aureus-infected human bone mesenchymal stem cells by sponging microRNA miR-29b-3p

Osteomyelitis (OM) is an orthopedic disease caused by bone infections in the bone cortex, bone marrow, periosteum, and surrounding soft tissues. Recent studies have implicated non-coding RNAs (ncRNAs) in the development of OM. However, little is known about the role of ncRNAs in the osteogenic differentiation during bone infection. In the present study, we investigated the role of KCNQ1OT1/miR-29b-3p axis in osteogenic differentiation in staphylococcus aureus (SpA)-infected human bone mesenchymal stem cells (hBMSCs). We first examined the expression of lncRNA KCNQ1OT1 and miR-29b-3p in the serum samples of OM patients and healthy controls. We also infected hBMSCs with different concentrations of SpA and studied the osteogenic differentiation after infection. Our results revealed that KCNQ1OT1 was downregulated while miR-29b-3p was upregulated in the serum samples of OM patients, as well as in SpA-infected hBMSCs. Overexpression of KCNQ1OT1 ameliorated the damage in hBMSCs caused by SpA infection. KCNQ1OT1 could support hBMSCs osteogenic differentiation by enhancing ALP activity, alizarin red S accumulation, expressions of osteogenic markers, and attenuating inflammatory responses after SpA infection. We further showed that miR-29b-3p was a downstream target of KCNQ1OT1, mediating the osteogenic differentiation of hBMSCs during SpA infection. Our data suggest that KCNQ1OT1 could ameliorate the SpA-induced suppression of osteogenic differentiation in hBMSCs by sponging miR-29b-3p. Modulating KCNQ1OT1 expression may serve as a strategy to ameliorate osteomyelitis.</p

The effect of DHA on GPDH activity and gene expressions of yellow croaker adipocytes during differentiation.

<p>The confluent cells were exposed to the differentiation medium without the lipid mixture but supplemented with 0, 50, 100 or 200 µmol/L DHA for 6 days and evaluated by GPDH, the indicator of cell differentiation. The gene expression levels were determined by quantitative Real-Time PCR. Data were analyzed by using 2^−ΔΔCt<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048069#pone.0048069-Livak1" target="_blank">[87]</a> and are referred to the control treatment (DHA = 0) using β-actin as a control. Data are means ± SEM, n = 3. Different letters indicate significant differences at <i>P</i><0.05. ATGL =  adipose triglyceride lipase, FAS = fatty acid synthase, LPL = lipoprotein lipase, PPAR = proliferators-activated receptor α, γ.</p

Micrographs of large yellow croaker adipocytes differentiated in culture.

<p>The cells were induced to differentiate into adipocytes and stained with oil red O at (A) day 3, (B) day 6 and (C) day 9 (×200) after induction. Electron micrographs of yellow croaker preadipocytes differentiated in culture at (D) day 3, (E) day 6 and (F) day 9 after induction. Bars: D, E = 1 µm, F = 2 µm. Arrows points to lipid droplets.</p

Targeted Proteomic Analysis of Small GTPases in Murine Adipogenesis

Small GTPases are essential signaling molecules for regulating glucose uptake in adipose tissues upon insulin stimulation, and this regulation maintains an appropriate range of glycemia. The involvement of small GTPases in adipogenesis, however, has not been systemically investigated. In this study, we applied a high-throughput scheduled multiple-reaction monitoring (MRM) method, along with the use of synthetic stable isotope-labeled peptides, to identify differentially expressed small GTPase proteins during adipogenesis of cultured murine cells. We were able to quantify the relative levels of expression of 55 and 49 small GTPases accompanied by adipogenic differentiation in 3T3-L1 and C3H10T1/2 cells, respectively. When compared with analysis conducted in the data-dependent acquisition (DDA) mode, the MRM-based proteomic platform substantially increased the coverage of the small GTPase proteome. Western blot analysis further corroborated the MRM quantification results for selected small GTPases. Interestingly, overall a significant number of small GTPases were down-regulated during adipogenesis. Among them, the expression levels of Rab32 protein were consistently lower in differentiated adipocytes than the corresponding undifferentiated precursors in both cell lines. Overexpression of Rab32 in 3T3-L1 and C3H10T1/2 cells prior to adipogenesis induction suppressed their differentiation. Together, this is the first comprehensive analysis of the alterations in small GTPase proteome during adipogenesis, and we reveal a previously unrecognized role of Rab32 in adipogenic differentiation

The effect of TNFα on GPDH activity and gene expression of large yellow croaker adipocytes during differentiation.

<p>The confluent cells were exposed to the differentiation medium without the lipid mixture but supplemented with 0, 1, 10 or 100 ng/ml hrTNFα for 6 days and evaluated by GPDH, the indicator of cell differentiation. The gene expression levels were determined by quantitative Real-Time PCR. Data were analyzed by using 2^−ΔΔCt<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048069#pone.0048069-Livak1" target="_blank">[87]</a> and are referred to the control treatment (TNFα = 0) using β-actin as a control. Data are means ± SEM, n = 3. Different letters indicate significant differences at <i>P</i><0.05. ATGL =  adipose triglyceride lipase, FAS = fatty acid synthase, LPL = lipoprotein lipase, PPAR  = proliferators-activated receptor α, γ.</p

GPDH activity of large yellow croaker adipocytes induced by different medium¹.

<p>The confluent cells were induced by growth medium, growth medium + hormones (containing 10 µg/ml insulin, 0.25 µM dexamethasone, and 0.5 mM IBMX), growth medium + lipid mixture (containing 45 µg/mL cholesterol, 100 µg/mL cod liver oil fatty acids) or growth medium + hormones +lipid mixture for 6 days and evaluated by GPDH, the indicator of cell differentiation.</p>1<p>Values are mean ± SEM obtained from six wells. Different letters indicate significant differences at <i>P</i><0.05.</p

The effect of insulin on GPDH activity and gene expressions of large yellow croaker adipocytes during differentiation.

<p>The confluent cells were exposed to the differentiation medium without the lipid mixture but supplemented with 0, 0.5, 5 or 50 µg/ml insulin for 6 days and evaluated by GPDH, the indicator of cell differentiation. The gene expression levels were determined by quantitative Real-Time PCR. Data were analyzed by using 2^−ΔΔCt<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048069#pone.0048069-Livak1" target="_blank">[87]</a> and are referred to the control treatment (insulin  = 0) using β-actin as a control. Data are means ± SEM, n = 3. Different letters indicate significant differences at <i>P</i><0.05. ATGL =  adipose triglyceride lipase, FAS = fatty acid synthase, LPL = lipoprotein lipase, PPAR  = proliferators-activated receptor α, γ.</p