Modulation of MKP-2 expression in adipose tissue by HFD feeding and in macrophages by FFA stimulation.

<p><i>(A)</i> WAT was isolated from mice fed with either a chow diet (NC) or a high-fat diet (HFD) for 8 week (4–5 mice per group). SVCs and adipocytes were isolated from the WAT to examine the expression of <i>Mkp-2</i> by quantitative real-time PCR (qRT-PCR). <i>(B) Mkp-2</i> expression was determined by qRT-PCR in RAW264.7 in response to 50μM palmitate stimulation. <i>(C)</i> MKP-2 protein expression was examined by western blot analysis in RAW264.7 in response to 50μM palmitate stimulation. The level of β-actin was examined as loading control. The intensity of the MKP-2 bands on the blots was quantified by ImageJ showing mean ± SEM from three independent experiments normalized to β-actin. <i>(D)</i> RAW264.7 cells were transfected with pcDNA3.1 or pcDNA3.1-MKP-2 plasmids together with AP-1 reporter plasmid and <i>Renilla</i> luciferase reporter vector. Dual luciferase assay was performed to assess AP-1 activity in response to 50μM FFA stimulation for 6h. The data shown are representative of three independent experiments with similar results. Data are presented as mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001.</p

Overexpression of MKP-2 enhances macrophage M2 activation.

<p><i>(A)</i> Control and MKP-2 overexpressing RAW264.7 cells were stimulated with 20 ng/mL IL-4 for 12h and total RNA was extracted to analyze the expression of M2 markers by qRT-PCR. <i>(B)</i> Control and MKP-2 overexpressing RAW264.7 cells were stimulated with 20 ng/mL IL-13 for 12h and total RNA was extracted to analyze expression of M2 markers by qRT-PCR. The data shown are representative of three independent experiments with similar results. Data are presented as mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001.</p

Overexpression of MKP-2 inhibits macrophage M1 activation and JNK/p38 activation.

<p>Control and MKP-2 overexpressing RAW264.7 cells were primed with 20 ng/mL IFN-γ for 12h followed by stimulation with 100 ng/mL LPS for another 12h. Culture supernatants were harvested for ELISA to detect IL-6 and TNF-α production <i>(A)</i>, and total RNA was extracted for qRT-PCR of <i>Il-12p40 (B)</i>. Cell lysates were harvested at 0 hour (h), 0.5h, 1h and 3h after M1 activator stimulation to determine the expression and activation of ERK, JNK, and p38 by western blot analysis <i>(C)</i>. <i>(D)</i> The intensity of phospho (p)-ERK, p-JNK, and p-p38 bands on the blots were quantified by ImageJ showing mean ± SEM from three independent experiments normalized to total ERK, JNK, and p38. Data are presented as mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001.</p

Overexpression of MKP-2 in macrophages inhibits inflammatory cytokine production during macrophage-adipocyte interaction.

<p><i>(A-B)</i> Control or MKP-2 overexpressing RAW264.7 (1x10⁵) and differentiated 3T3-L1 cells were cultured either alone or together and stimulated with or without 750μM palmitate overnight. IL-6, TNF-α, and MCP-1 levels in the supernatants were determined by ELISA. Cytokine production was normalized to total protein of the cell lysates. mRNA of each sample was harvested to examine <i>Mkp-2</i> expression by qRT-PCR. Ct, control; Co, co-culture. <i>(C)</i> Control and MKP-2 overexpressing RAW264.7 cells were treated with conditioned medium (CM) derived from 3T3-L1 adipocytes treated with BSA (CM-B) or 750μM FFA (CM-F) for 24 h. Regular media containing BSA (RM-B) or 750μM FFA (RM-F) were used as control. Supernatants were harvested for ELISA analysis. <i>(D)</i> CM derived from vector transfected or MKP-2 overexpressing RAW264.7 treated with BSA or 750μM FFA were used to treat 3T3-L1 adipocytes for 24 h. Regular media containing BSA or 750μM FFA were used as control. Supernatants were harvested for ELISA analysis. RM, regular media; CM-pcDNA3.1, conditioned media derived from pcDNA3.1-transfected RAW264.7; CM-MKP-2, conditioned media derived from MKP-2 transfected RAW264.7. Data shown are representative of three independent experiments with similar results. Data are presented as mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001.</p

Inhibition of JNK and p38 in macrophages suppressed inflammatory cytokine expression in macrophages and in macrophage-adipocyte interaction.

<p><i>(A)</i> RAW264.7 cells were stimulated with IFN-γ plus LPS in the presence of JNK-specific inhibitor SP600125 (20μM) (Sigma) or p38-specific inhibitor SB23580 (20μM) for the indicated time points. DMSO was used as vehicle control. The expression and activation of JNK or ATF2 were determined by western blot analysis. RAW264.7 cells were pretreated with SP600125 (20μM) followed by stimulation with M1 <i>(B)</i> or M2 <i>(C)</i> activation condition. The expression of M1 or M2 markers was determined. RAW264.7 cells were pretreated with SB23580 (20μM) followed by stimulation with M1 <i>(D)</i> or M2 <i>(E)</i> activation condition. The expression of M1 or M2 genes was analyzed. <i>(F)</i> Control and MKP-2 overexpressing RAW264.7 cells were pretreated with SP600125 (20μM) or SB23580 (20μM) or both for 1h before co-culture with differentiated 3T3-L1 adipocytes for 24h. The concentrations of MCP-1, IL-6, and TNF-α in culture supernatants were determined by ELISA. Cytokines production was normalized to total protein of the cell lysates. Ct, control; Co, co-culture. Data shown are representative of three independent experiments with similar results. Data are presented as mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001.</p

DataSheet_1_Establishment of a circular RNA regulatory stemness-related gene pair signature for predicting prognosis and therapeutic response in colorectal cancer.xlsx

BackgroundColorectal cancer (CRC) is a common malignant tumor of the digestive tract with a poor prognosis. Cancer stem cells (CSCs) affect disease outcomes and treatment responses in CRC. We developed a circular RNA (circRNA) regulatory stemness-related gene pair (CRSRGP) signature to predict CRC patient prognosis and treatment effects.MethodsThe circRNA, miRNA, and mRNA expression profiles and clinical information of CRC patients were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases. CRSRGPs were established based on stemness-related genes in the competing endogenous RNA (ceRNA) network. A CRSRGP signature was generated using the least absolute shrinkage and selection operator (Lasso) and Cox regression analysis of TCGA training set. The prognosis was predicted by generating a nomogram integrating the CRSRGP signature and clinicopathologic features. The model was validated in an external validation set (GSE17536). The antitumor drug sensitivity and immunotherapy responses of CRC patients in the high-risk group (HRG) and low-risk group (LRG) were evaluated by the pRRophetic algorithm and immune checkpoint analysis.ResultsWe established an 18-CRSRGP signature to predict the prognosis and treatment responses of CRC patients. In the training and external validation sets, risk scores were used to categorize CRC patients into the HRG and LRG. The Kaplan–Meier analysis showed a poor prognosis for patients in the HRG and that subgroups with different clinical characteristics had significantly different prognoses. A multivariate Cox analysis revealed that the CRSRGP signature was an independent prognostic factor. The nomogram integrating clinical features and the CRSRGP signature efficiently predicted CRC patient prognosis, outperformed the current TNM staging system, and had improved practical clinical value. Anticancer drug sensitivity predictions revealed that the tumors of patients in the HRG were more sensitive to pazopanib, sunitinib, gemcitabine, lapatinib, and cyclopamine. Analysis of immune checkpoint markers demonstrated that patients in the HRG were more likely to benefit from immunotherapy.ConclusionAn efficient, reliable tool for evaluating CRC patient prognosis and treatment response was established based on the 18-CRSRGP signature and nomogram.</p

Overexpression of MKP-2 inhibits TNF-α expression and JNK activation in macrophages in response to FFA.

<p><i>(A)</i> MKP-2 expression in MKP-2 stably transfected RAW264.7 was examined by qRT-PCR and western blot. <i>(B)</i> Control and MKP-2 overexpressing RAW264.7 cells were stimulated with indicated concentrations of palmitate for 24 h, and culture supernatants were harvested to measure TNF-α production by ELISA. <i>(C)</i> Control and MKP-2 overexpressing RAW264.7 cells were stimulated with 750μM palmitate for the indicated time points and cell lysates were prepared to examine JNK expression and activation. <i>(D)</i> The intensity of the p-JNK bands on the blots was quantified by ImageJ showing mean ± SEM from three independent experiments normalized to total JNK. Data are presented as mean ± SEM. *, p<0.05; **, p<0.01; ***, p<0.001.</p

DataSheet_1_Mendelian randomization study shows a causal effect of asthma on epilepsy risk.xlsx

ObjectiveThe relationship between asthma and epilepsy in observational studies is controversial. The purpose of this Mendelian randomization (MR) study is to investigate whether asthma causally contributes to epilepsy susceptibility.MethodsIndependent genetic variants strongly (PResultsUsing the inverse-variance weighted approach, genetic predisposition to asthma was associated with an elevated risk of epilepsy in the discovery stage (ILAEC: odds ratio [OR]=1.112, 95% confidence intervals [CI]= 1.023-1.209, P = 0.012), but not verified in the replication stage (FinnGen: OR=1.021, 95%CI= 0.896–1.163, P =0.753). However, a further meta-analysis of both ILAEC and FinnGen showed a similar result (OR=1.085, 95% CI: 1.012-1.164, P = 0.022). There were no causal associations between the age onset of asthma and epilepsy. Sensitivity analyses yielded consistent causal estimates.ConclusionThe present MR study suggests that asthma is associated with an increased risk of epilepsy independent of the age onset of asthma. Further studies are warranted to explain the underlying mechanisms of this association.</p

DataSheet_2_Mendelian randomization study shows a causal effect of asthma on epilepsy risk.docx

ObjectiveThe relationship between asthma and epilepsy in observational studies is controversial. The purpose of this Mendelian randomization (MR) study is to investigate whether asthma causally contributes to epilepsy susceptibility.MethodsIndependent genetic variants strongly (PResultsUsing the inverse-variance weighted approach, genetic predisposition to asthma was associated with an elevated risk of epilepsy in the discovery stage (ILAEC: odds ratio [OR]=1.112, 95% confidence intervals [CI]= 1.023-1.209, P = 0.012), but not verified in the replication stage (FinnGen: OR=1.021, 95%CI= 0.896–1.163, P =0.753). However, a further meta-analysis of both ILAEC and FinnGen showed a similar result (OR=1.085, 95% CI: 1.012-1.164, P = 0.022). There were no causal associations between the age onset of asthma and epilepsy. Sensitivity analyses yielded consistent causal estimates.ConclusionThe present MR study suggests that asthma is associated with an increased risk of epilepsy independent of the age onset of asthma. Further studies are warranted to explain the underlying mechanisms of this association.</p

Imaging situation for an unsuitable plan.

(a) Inappropriate working distance results in the inability to achieve a detection accuracy of 0.05mm. (b) Due to factors such as working distance or focal length, as well as the imaging chip of the lens, the precision cannot reach 0.05mm.</p